In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A 2 pm 3 3 A 2 pm 3 (A 2 pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala 4 3 A 2 pm 3 cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala 4 . Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt cd1 and ldt cd2 , were inactivated. The inactivation of either ldt cd1 or ldt cd2 significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt cd1 -and ldt cd2 -encoded proteins have a redundant L,D-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,D-transpeptidation pathway in C. difficile.Clostridium difficile, a Gram-positive spore-forming bacterium, is the major cause of intestinal diseases associated with antibiotic therapy such as ampicillin, clindamycin, and cephalosporins, which disrupt the barrier intestinal flora and allow C. difficile colonization (1, 2). Clinical manifestations range from asymptomatic colonization or mild diarrhea to pseudomembranous colitis (3). The main virulence factors have been identified as toxin A and B (4). Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new highly virulent strain (BI/NAP1/027) of C. difficile.Antibiotic treatment of C. difficile-associated disease requires metronidazole or vancomycin therapy.Peptidoglycan ( (9), which were originally detected in Enterococcus faecium (Ldt fm ) (10) and then in other Gram-positive bacteria (11,12), in mycobacteria (13-15), and in Escherichia coli (16,17). Ldts use acyl donors containing a tetrapeptide stem (9) and were consequently expected to confer resistance to ␤-lactams (10, 18).Another possible variation of the PG structure is the occurrence of N-deacetylation or O-acetylation of glycan strands, either on GlcNAc or on MurNAc residues (19,20). N-Deacetylation in Listeria monocytogenes (21) or Streptococcus pneumoniae (22) and O-acetylation in Staphylococcus aureus have been linked to lysozyme resistance (23).The PGs of C. difficile should have some specificities regarding the effect of antibiotics inhibiting PG biosynthesis; C. difficile, although susceptible to ␤-lactams, exhibits higher minimal inhibitory concentrations than in oth...

Analysis of the genetic locus encompassing a cell wall polysaccharide (CWPS) biosynthesis operon of eight strains of Lactococcus lactis, identified as belonging to the same CWPS type C genotype, revealed the presence of a variable region among the strains examined. The results allowed the identification of five subgroups of the C type named subtypes C1 to C5. This variable region contains genes encoding glycosyltransferases that display low or no sequence homology between the subgroups. In this study, we purified an acidic polysaccharide from the cell wall of L. lactis 3107 (subtype C2) and confirmed that it is structurally different from the previously established CWPS of subtype C1 L. lactis MG1363. The CWPS of L. lactis 3107 is composed of pentasaccharide repeating units linked by phosphodiester bonds with the structure 6-α-Glc-3-β-Galf-3-β-GlcNAc-2-β-Galf-6-α-GlcNAc-1-P. Combinations of genes from the variable region of subtype C2 were introduced into a mutant of subtype C1 L. lactis NZ9000 deficient in CWPS biosynthesis. The resulting recombinant mutant synthesized a polysaccharide with a composition characteristic of that of subtype C2 L. lactis 3107 and not wild-type C1 L. lactis NZ9000. By challenging the recombinant mutant with various lactococcal phages, we demonstrated that CWPS is the host cell surface receptor of tested bacteriophages of both the P335 and 936 groups and that differences between the CWPS structures play a crucial role in determining phage host range.

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Peptidoglycans provide bacterial cell walls with mechanical strength. The spatial organization of peptidoglycan has previously been difficult to study. Here, atomic force microscopy, together with cells carrying mutations in cell-wall polysaccharides, has allowed an in-depth study of these molecules.

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has L,D-carboxypeptidase activity and is involved in peptidoglycan maturation.Peptidoglycan is the major component of the gram-positive bacterial cell wall and ensures its rigidity and stability. Although its basic structure is characteristic of a given bacterial species, peptidoglycan is in a dynamic state throughout the bacterial life span, and its structure is the result of complex biosynthetic, maturation, and degradation reactions (11). Structural analysis of the peptidoglycan constituent muropeptide is a powerful method that allowed elucidation of the roles of biosynthesis enzymes involved in the design of cell wall architecture (3) and to characterize changes in peptidoglycan structure leading to antibiotic resistance (1,8,16). Also, the technique allowed revelation of peptidoglycan covalent modifications, such as O-acetylation or de-N-acetylation, which could play essential roles in the control of the activities of exogenous (25) and endogenous (17) cell wall-degrading enzymes.Lactococcus lactis is the model gram-positive lactic acid bacterium. Its peptidoglycan hydrolase complement was previously characterized (7,12,13,22). Bacterial peptidoglycan hydrolases are involved in different cellular functions during growth, such as cell separation, cell wall turnover, and cell wall expansion (21). Their activities can also lead to bacterial autolysis by hydrolysis of the protective cell wall peptidoglycan. Since these potentially lethal enzymes are synthesized during bacterial growth, their activities should be controlled. As mentioned above, covalent structural modification of peptidoglycan is one of the proposed mechanisms that could control peptidoglycan hydrolase activity (17, 21). Thus, the analysis of the L. lactis peptidoglycan structure constitutes the basis for further studies of the mechanisms that regulate synthesis and degradation of the L. lactis cell wall. Earlier studies revealed that L. lactis (formerly Streptococccus lactis) has A4␣-type peptidoglycan, with a monomer primary structure (GlcNAcMurNAc-L-Ala-␣-D-Glu-L-Lys-D-Ala) and a D-Asp in the interpeptide bridge, attached to the ε-amino group of Lys (19). In this study, we achieved detailed analysis of the muropeptide composition of Lactococcus lactis. Also, using the method developed, we identified an L,D-carboxypeptidase in L. lactis involved in peptidoglycan maturation.Muropeptide composition of L. lactis MG1363. Lactococcus lactis subsp. cremoris MG1363 was grown on M17 medium conta...

SummaryThe microbial environment influence animal physiology. However, the underlying molecular mechanisms of such functional interactions are largely undefined. Previously, we showed that upon chronic undernutrition, strains of Lactobacillus plantarum, a major commensal partner of Drosophila, promote host juvenile growth and maturation partly via enhanced expression of intestinal peptidases. By screening a transposon insertion library of Lactobacillus plantarum in gnotobiotic Drosophila larvae, we identify a bacterial cell wall modifying machinery encoded by the pbpX2-dlt operon that is critical to enhance host digestive capabilities and promote animal growth and maturation. Deletion of this operon leads to bacterial cell wall alteration with a complete loss of teichoic acids D-alanylation. We show that L. plantarum cell walls bearing D-alanylated teichoic acids are directly sensed by Drosophila enterocytes to ensure optimal intestinal peptidase expression and activity, juvenile growth and maturation upon chronic undernutrition. We thus conclude that besides peptidoglycan, teichoic acids modifications participate in the host-commensal bacteria molecular dialogue occurring in the intestine.