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 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.

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.

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.

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...

Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The ⌬EF_0783 mutant and ⌬EF_0783 ⌬EF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ⌬EF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ⌬EF_0783 and ⌬EF_0783 ⌬EF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis.Enterococcus faecalis is intrinsically not as virulent as other gram-positive organisms, such as Staphylococcus aureus, pneumococci, or group A streptococci. However, E. faecalis emerges as an opportunistic pathogen and one of the leading causes of hospital-acquired infections, such as urinary tract, surgical wound, abdominal, pelvic, and neonatal infections in the United States and northern Europe (26). Little is known about its virulence potential. This lack of information can be partly attributed to the fact that E. faecalis, which normally grows as a commensal organism in the gut, possesses very subtle virulence traits that are not easily identified (27). Several surface proteins, enzymes, and capsular polysaccharides likely involved in virulence and the ability of E. faecalis to survive inside polymorphonuclear leukocytes (36) and macrophages (25) may contribute to its pathogenicity. Besides, intrinsic physiological properties of E. faecalis, such as its exceptional resistance to harsh conditions (37) and its inherent antibiotic resistance (27), may also provide an advantage during the infection process. In order to survive and colonize the host, bacteria must overcome the constitutive or innate defense system and the host's phagocytic response to achieve infection. One of the most important and widespread compounds of the constitutive defense s...