Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan.IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus Lactococcus lactis, a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the L. lactis cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci.

Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.

The recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five-fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry-based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms.

Maternal high-protein or high-prebiotic-fiber diets affect maternal milk composition and gut microbiota in rat dams and their offspring.

OBJECTIVE: Maternal gut microbiota and milk composition could modify offspring microbiota and therefore disease susceptibility. The effect of maternal high-protein (HP) or prebiotic diets on maternal milk composition and gut microbiota in rat dams and offspring was examined. METHODS: Wistar rat dams were fed a control, HP (40% wt/wt), or high-prebiotic-fiber (21.6% wt/wt) (HF) diet throughout pregnancy and lactation. Pups were challenged with a high-fat/sucrose diet from 14.5 to 22.5 weeks of age. Dam milk was analyzed for fat, protein, and oligosaccharides (OS). Fecal microbiota was analyzed in dams at parturition and 2 weeks post-partum and in offspring at 5 and 22 weeks along with cecal digesta at termination. RESULTS: Maternal milk differed only in OS content, each diet group being distinguishable. HF1 and HP1 offspring had decreased plasma lipopolysaccharide compared with C1. Offspring sex, maternal diet, and time (5 weeks vs. 22 weeks of age) affected the microbial groups examined. Bifidobacteria was higher in HF dams and offspring. CONCLUSIONS: Increasing protein or fiber content in maternal diet during pregnancy and lactation modifies milk OS content and gut microbiota of dams which may influence establishment of gut microbiota in offspring.

Unraveling the role of surface mucus-binding protein and pili in muco-adhesion of Lactococcus lactis.

Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100-200 nm) and long distances (up to 600-800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants.

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.

Identification and characterisation of water and alkali soluble oligosaccharides from hazelnut skin (Corylus avellana L.).

Hazelnut skins are a good example of agricultural by-product with the potential to become a valuable source of functional ingredients. In this work, the fibre from hazelnut skins was extracted by using water and alkali solution and characterised by a suite of analytical tools (MALDI-FTICR, nano LC-Chip-Q-ToF and gas chromatography). Over thirty complex free oligosaccharides, composed mainly of galacturonic acid and N-acetylgalactosamine, were characterised for the first time in the present study. Their concentration ranged between 16 and 34mg per g of extract. The oligosaccharides isolated from this agricultural by-product are mainly hexose oligosaccharides (potentially galacto-oligosaccharides,) and xyloglucans. The identified composition could justify the bioactive activity of the extracts, namely prebiotic activity, previously demonstrated.

Natural variability in bovine milk oligosaccharides from Danish Jersey and Holstein-Friesian breeds.

Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds, Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contains levels of fucosylated oligosaccharides significantly higher than that of Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid), suggesting it may therefore offer advantages in term of a wider array of bioactivities.

Identification and characterization of complex bioactive oligosaccharides in white and red wine by a combination of mass spectrometry and gas chromatography.

Over forty-five complex free oligosaccharides (of which several are novel) have been isolated and chemically characterized by gas chromatography and high resolution and high mass accuracy matrix-assisted laser desorption/ionization mass spectrometry (MALDI-FTICR MS) in red and white wines, Grignolino and Chardonnay, respectively. Oligosaccharides with a degree of polymerization between 3 and 14 were separated from simple monosaccharides and disaccharides by solid-phase extraction. The concentrations of free oligosaccharides were over 100 mg/L in both red and white wines. The free oligosaccharides-characterized for the first time in the present study-include hexose-oligosaccharides, xyloglucans, and arabinogalactans and may be the natural byproduct of the degradation of cell wall polysaccharides. The coupled gas chromatography and accurate mass spectrometry approach revealed an effective method to characterize and quantify complex functional oligosaccharides in both red and white wine.

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.

Variations of N-acetylation level of peptidoglycan do not influence persistence of Lactococcus lactis in the gastrointestinal tract.

The food-grade Gram-positive bacterium, Lactococcus lactis, is recognized as a potential candidate to deliver proteins of medical interest by mucosal routes. The ability of carrier bacteria to persist and/or to lyse in the gastrointestinal tract needs to be considered to design optimal carrier strains to deliver proteins of interest at the mucosal level. Meyrand et al. (2007) have previously characterized in L. lactis, a peptidoglycan (PG) N-acetylglucosamine deacetylase (PgdA), which activity on PG influences bacterial sensitivity to lysozyme. Inactivation of pgdA gene in this bacterium, led to fully acetylated PG, resulting in a lysozyme-sensitive phenotype, whereas pgdA overexpression led to an increased degree of PG deacetylation, resulting in a lysozyme-resistant phenotype (Meyrand et al., 2007). In order to determine whether variations in L. lactis resistance to host lysozyme may influence its persistence in the GIT and its ability to deliver heterologous proteins in situ, we constructed L. lactis strains with different de-N-acetylation levels and producing a model antigen (the human papillomavirus type-16 E7 protein) and we compared the pharmacokinetics properties of these recombinant strains with that of a wild-type strain producing the same antigen in the GIT of mice. Our results show that there was no correlation between survival, at the ileum level, of bacteria intragastrically administered in mice and bacteria sensitivity or resistance to lysozyme. In addition, analysis of the E7-specific immune response evoked by the three strains after mucosal administration in mice suggest that neither lysozyme-sensitive nor lysozyme-resistant phenotype in L. lactis enhances significantly the potential of this bacterium as mucosal delivery live vector. In conclusion, our results suggest that either pgdA inactivation or pgdA overexpression in L. lactis leading to different levels of PG deacetylation does not confer any advantage in the persistence of this bacterium in the GIT and its ability to enhance host immune responses induced by delivered antigen in situ.

Genetic features of resident biofilms determine attachment of Listeria monocytogenes.

Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.

Variations in the degree of D-Alanylation of teichoic acids in Lactococcus lactis alter resistance to cationic antimicrobials but have no effect on bacterial surface hydrophobicity and charge.

An increase of the degree of d-alanylation of teichoic acids in Lactococcus lactis resulted in a significant increase of bacterial resistance toward the cationic antimicrobials nisin and lysozyme, whereas the absence of D-alanylation led to a decreased resistance toward the same compounds. In contrast, the same variations of the D-alanylation degree did not modify bacterial cell surface charge and hydrophobicity. Bacterial adhesion to polystyrene and glass surfaces was not modified either.

Proteomic investigation of the adaptation of Lactococcus lactis to the mouse digestive tract.

Lactic acid bacteria are used on an industrial scale for the manufacturing of dairy products. It is now intended to develop novel applications of lactic acid bacteria that could be used as living vehicles for the targeting of antigens or therapeutics to the digestive mucosa. The aim of this study was to analyze the adaptations of Lactococcus lactis, a model lactic acid bacteria to the digestive tract and to identify functions required for colonization of the intestine. For this purpose, we combined gnotobiology with proteomics: axenic mice were colonized with a dairy L. lactis strain and the bacterial proteome was examined by 2-DE. As compared to cultures in broth, the proteome profile of bacteria grown in the intestine indicates the activation of metabolic pathways involved in various carbon sources assimilation and suggests the adoption of a mixed acids fermentative metabolism. We identified the product of the ywcC gene as essential for the colonization of the digestive tract and demonstrated that the corresponding gene product (YwcC) possesses a phosphogluconolactonase activity, suggesting an important role of the pentose phosphate pathway for the development of L. lactis in the digestive environment.

Peptidoglycan N-acetylglucosamine deacetylation decreases autolysis in Lactococcus lactis.

The gene xynD (renamed pgdA) of Lactococcus lactis IL1403 was shown to encode a peptidoglycan N-acetylglucosamine deacetylase. Inactivation of pgdA in L. lactis led to fully acetylated peptidoglycan, whereas cloning of pgdA on a multicopy plasmid vector resulted in an increased degree of peptidoglycan deacetylation, as shown by analysis of peptidoglycan constituent muropeptides. An increased amount of N-unsubstituted glucosamine residues in peptidoglycan resulted in a reduction of the rate of autolysis of L. lactis cells. The activity of the L. lactis major autolysin AcmA was tested on L. lactis cells or peptidoglycan with different degrees of de-N-acetylation. Deacetylated peptidoglycan exhibited decreased susceptibility to AcmA hydrolysis. This reduced susceptibility to AcmA did not result from reduced AcmA binding to peptidoglycan with an increasing degree of de-N-acetylation. In conclusion, enzymic N-acetylglucosamine deacetylation protects peptidoglycan from hydrolysis by the major autolysin AcmA in L. lactis cells, and this leads to decreased cellular autolysis.

Proteomic signature of Lactococcus lactis NCDO763 cultivated in milk.

We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment.