Over-production of exopolysaccharide by Lacticaseibacillus rhamnosus CNCM I-3690 strain cutbacks its beneficial effect on the host.

Most lactobacilli produce extracellular polysaccharides that are considered to contribute to the probiotic effect of many strains. Lacticaseibacillus rhamnosus CNCM I-3690 is an anti-inflammatory strain able to counterbalance gut barrier dysfunction. In this study ten spontaneous variants of CNCM I-3690 with different EPS-production were generated and characterized by their ropy phenotype, the quantification of the secreted EPS and genetic analysis. Amongst them, two were further analysed in vitro and in vivo: an EPS over-producer (7292) and a low-producer derivative of 7292 (7358, with similar EPS levels than the wild type (WT) strain). Our results showed that 7292 does not have anti-inflammatory profile in vitro, and lost the capacity to adhere to the colonic epithelial cells as well as the protective effect on the permeability. Finally, 7292 lost the protective effects of the WT strain in a murine model of gut dysfunction. Notably, strain 7292 was unable to stimulate goblet cell mucus production and colonic IL-10 production, all key features for the beneficial effect of the WT strain. Furthermore, transcriptome analysis of colonic samples from 7292-treated mice showed a down-regulation of anti-inflammatory genes. Altogether, our results point out that the increase of EPS production in CNCM I-3690 impairs its protective effects and highlight the importance of the correct EPS synthesis for the beneficial effects of this strain.

Characterisation of autolytic enzymes in Lactobacillus pentosus.

AIMS: To characterize autolysis and autolytic system of the lactic acid bacterium Lactobacillus pentosus. METHODS AND RESULTS: Autolysis of nine Lact. pentosus strains was evaluated in buffer solution. Their peptidoglycan hydrolase profiles were examined by renaturing SDS-PAGE and revealed two major activity bands at 58 and 112 kDa. Specificity analysis indicated the presence of at least two different types of peptidoglycan hydrolase activities in Lact. pentosus 1091. CONCLUSIONS: Autolysis of Lact. pentosus was shown to be strain dependent and involvement of at least two different autolysins was evidenced. SIGNIFICANCE AND IMPACT OF THE STUDY: The autolytic system of Lact. pentosus was characterized for the first time and the data obtained could be used in the selection of strains of technological interest.

Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum.

A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.

Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE.

The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain.

The gene encoding Mur1, a Streptococcus thermophilus peptidoglycan hydrolase, was cloned by homology with acmA, the Lactococcus lactis major autolysin gene. Mur1 is a 24.7-kDa protein endowed with a putative signal peptide. Sequence analysis evidenced that Mur1 encompasses exactly the AcmA region containing the catalytic domain, but lacks the one containing amino acid repeats involved in cell wall binding. Mur1 appears to be expressed and cell-associated in S. thermophilus, as revealed by immunoblot analysis. These results suggest that the cell wall attachment mode of Mur1 differs from that of most peptidoglycan hydrolases described so far.

Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions.

The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.

The Streptococcus thermophilus autolytic phenotype results from a leaky prophage.

Streptococcus thermophilus autolytic strains are characterized by a typical bell-shaped growth curve when grown under appropriate conditions. The cellular mechanisms involved in the triggering of lysis and the bacteriolytic activities of these strains were investigated in this study. Lactose depletion and organic solvents (ethanol, methanol, and chloroform) were shown to trigger a premature and immediate lysis of M17 exponentially growing cells. These factors and compounds are suspected to act by altering the cell envelope properties, causing either the permeabilization (organic solvents) or the depolarization (lactose depletion) of the cytoplasmic membrane. The autolytic character was shown to be associated with lysogeny. Phage particles, most of which were defective, were observed in the culture supernatants after both mitomycin C-induced and spontaneous lysis. By renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a bacteriolytic activity was detected at 31 kDa exclusively in the autolytic strains. This enzyme was detected during both growth and spontaneous lysis with the same intensity. We have shown that it was prophage encoded and homologous to the endolysin Lyt51 of the streptococcal temperate bacteriophage phi01205 (M. Sheehan, E. Stanley, G. F. Fitzgerald, and D. van Sinderen, Appl. Environ. Microbiol. 65:569-577, 1999). It appears from our results that the autolytic properties are conferred to the S. thermophilus strains by a leaky prophage but do not result from massive prophage induction. More specifically, we propose that phagic genes are constitutively expressed in almost all the cells at a low and nonlethal level and that lysis is controlled and achieved by the prophage-encoded lysis proteins.

Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene.

Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli. This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis. One of them, cspB, was cloned and sequenced. It encodes a 66-residue protein which possesses 60% sequence identity with E. coli CspA. Following a cold shock from 30 to 15 degrees C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization. In addition, induction of cspB-directed beta-galactosidase activity was observed. These results indicate that the L. lactis cspB gene is cold shock inducible.

Gene cloning and characterization of PepC, a cysteine aminopeptidase from Streptococcus thermophilus, with sequence similarity to the eucaryotic bleomycin hydrolase.

Streptococcus thermophilus CNRZ 302 contains at least three general aminopeptidases able to hydrolyze Phe-beta-naphthylamide substrate. The gene encoding one of these aminopeptidases was cloned from a total DNA library of S. thermophilus CNRZ 302 constructed in Escherichia coli TG1 using pBluescript plasmid. The wild-type TG1 strain, although not deficient in aminopeptidase activity, is unable to hydrolyze the substrate Phe-beta-naphthylamide, and thus the library could be screened with an enzymic plate assay using this substrate. One clone was selected which was shown to express an aminopeptidase, identified as a PepC-like enzyme on the basis of cross-reactivity with polyclonal antibodies directed against the lactococcal PepC cysteine aminopeptidase. The gene was further subcloned and sequenced. A complete open reading frame coding for a 445-residue (50414 Da) polypeptide was identified. 70% identity was found between the deduced amino acid sequence and the sequence of PepC from Lactococcus lactis subspecies cremoris, confirming the identity of the cloned gene. High sequence similarity (38% identity) was also found with an eucaryotic enzyme, bleomycin hydrolase. In addition, the predicted amino acid sequence of the streptococcal PepC showed a region of strong similarity to the active site of cysteine proteinases with conservation of the residues involved in the catalytic site. The product of the cloned pepC gene was overproduced in E. coli and was purified from a cellular extract. Purification to homogeneity was achieved by two-step ion-exchange chromatography. Biochemical characterization of the pure recombinant enzyme confirms that the cloned peptidase is a thiol aminopeptidase possessing a broad specificity. The enzyme has a molecular mass of 300 kDa suggesting an hexameric structure. On the basis of sequence similarities as well as common biochemical and enzymic properties, the bacterial PepC-type enzymes and the eucaryotic bleomycin hydrolase constitute a new family of thiol aminopeptidases among the cysteine peptidases.

Crystallization and preliminary X-ray analysis of PepC, a thiol aminopeptidase from Lactoccocus lactis homologous to bleomycin hydrolase.

Crystals of the recombinant thiol aminopeptidase PepC, from Lactoccocus lactis, have been obtained using the hanging-drop method of vapor diffusion from ammonium sulfate solutions. Crystals are rhombohedral, the space group is R32, a = 175.2 A, c = 94.5 A (hexagonal setting). The asymmetric unit probably contains one monomer of a hexameric molecule-arrangement of 300 kDa which exhibits the crystallographic point group of symmetry 32. The crystals diffract to at least 3 A resolution.

Design of (omega-N-(O-acyl)hydroxy amid) aminodicarboxylic acid pyrrolidides as potent inhibitors of proline-specific peptidases.

A novel class of competitive, acylating inhibitors for the proline-specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N-terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting Ki values in the microM range. Boc-protected (omega-N-hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching Ki values of 30 nM to 60 microM. Additionally, alpha-N-BOC-(omega-N-hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time-dependent reaction.

Cloning and sequencing of pepC, a cysteine aminopeptidase gene from Lactococcus lactis subsp. cremoris AM2.

A gene coding for an aminopeptidase (PepC) from Lactococcus lactis subsp. cremoris AM2 was cloned by complementation of an Escherichia coli mutant lacking aminopeptidase activity. The nucleotide sequence was determined. A portion of the predicted amino acid sequence of PepC (436 amino acids) showed strong homology to the active site of cysteine proteases. No signal sequence was found, indicating an intracellular location of the enzyme.

Identification of the active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The active site serine of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse-phase-HPLC. The main radiolabeled peptide was sequenced. Ser-348, in the following sequence, Gly-Lys-Ser-Tyr-Leu-Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X-prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G-X-S-Y-X-G, where X is a non-conserved amino acid.

Localization of peptidases in lactococci.

The localization of two aminopeptidases, an X-prolyl-dipeptidyl aminopeptidase, an endopeptidase, and a tripeptidase in Lactococcus lactis was studied. Polyclonal antibodies raised against each purified peptidase are specific and do not cross-react with other peptidases. Experiments were performed by immunoblotting after cell fractionation and by electron microscopy of immunogold-labeled peptidases. All peptidases were found to be intracellular. However, immunogold studies showed a peripheral labeling of the X-prolyl-dipeptidyl aminopeptidase, the tripeptidase, and the endopeptidase. This peripheral location was further supported by the detection of these three enzymes in cell membrane fractions in which none of the two aminopeptidases was present.