Florence Valence

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFα, and IL-1β were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA–DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.

Development and Application of a upp-Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM

ABSTRACT In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organisms survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp-encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the Δupp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus. The resulting ΔslpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus.

Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains

Intracellular peptidases of Lactobacillus helveticus may play a major role in the proteolysis of Swiss cheeses, provided that they are released through bacterial lysis. Experimental Swiss cheeses were manufactured on a small scale from thermized and microfiltered milk using as starters (in addition to Streptococcus thermophilus and Propionibacterium freudenreichii) one of two Lb. helveticus strains, ITGLH1 and ITGLH77, which undergo lysis to different extents in vitro. All the cheeses were biochemically identical after pressing. The viability of Lb. helveticus ITGLH1 and ITGLH77 decreased to a similar extent (96-98%) while in the cold room, but the concomitant release of intracellular lactate dehydrogenase in cheeses made with strain ITGLH1 was 5-7-fold that in cheeses made with ITGLH77. Protein profiles and immunoblot detection of the dipeptidase PepD confirmed a greater degree of lysis of the ITGLH1 strain. Free active peptidases were detected in aqueous extracts of cheese for both strains, and proteolysis occurred principally in the warm room. Reversed-phase HPLC revealed a more extensive peptide hydrolysis for ITGLH1, which was confirmed by the greater release of free NH2 groups (+33%) and free amino acids (+75%) compared with ITGLH77. As the intracellular peptidase activities of ITGLH1 and ITGLH77 have previously been shown to be similar, our results indicated that the extent of lysis of Lb. helveticus could have a direct impact on the degree of proteolysis in Swiss cheeses.

Autolysis of Lactobacillus helveticus and Propionibacterium freudenreichii in Swiss cheeses: first evidence by using species-specific lysis markers

Lactobacillus helveticus and Propionibacterium freudenreichii are essential starters in Swiss cheesemaking and the release of their intracellular enzymes through autolysis could significantly influence ripening. To provide evidence of this lysis, cheese made from microfiltered thermized milk inoculated with Lb. helveticus ITGLH77, Prop. freudenreichii ITGP23 and a commercial Streptococcus thermophilus was assayed. Starter viability was determined and lysis was monitored during ripening by protein analysis with SDS-PAGE of aqueous cheese extracts and by immunoblot detection of intracellular proteins: dipeptidase (PepD) for Lb. helveticus and methylmalonyl coenzyme A mutase for Prop. freudenreichii . We verified that the species specificity of these lysis markers was towards the cytoplasms of all the species currently used in Swiss cheese. Lb. helveticus exhibited an almost complete loss of viability (99·9%) from the beginning of ripening in the cold room; concomitantly PepD appeared in the cheese extracts and was detected until the end of ripening. Damaged Lb. helveticus cells were also visualized by scanning electron microscopy. In addition, free PepD was also successfully detected in commercial Swiss-related cheeses. All these results clearly demonstrated the autolysis of Lb. helveticus in Swiss cheese. Prop. freudenreichii ITGP23 grew during warm room ripening and no loss of viability was detected after maximal growth (10 9 cfu/g cheese). Free methylmalonyl-coenzyme A mutase was detected at the end of ripening during cold storage, when the cheese extracts were concentrated 20-fold, demonstrating that the autolysis of Prop. freudenreichii was tardy and limited.

Electrophoretic pattern of peptidoglycan hydrolases, a new tool for bacterial species identification : application to 10 Lactobacillus species

Lactobacilli have been used as industrial starters for a long time, but in many cases their phenotypic identification is still neither easy nor reliable. Previously we observed that the cell wall peptidoglycan hydrolases of Lactobacillus helveticus were highly conserved enzymes; the aim of the present work was to determine whether peptidoglycan hydrolase patterns obtained by renaturing SDS-PAGE could be of interest in the identification of lactobacilli species. For that purpose, the peptidoglycan hydrolase patterns of 94 strains of lactobacilli belonging to 10 different species were determined; most of the species studied are used either in dairy, meat, bakery or vegetable fermentations: L. helveticus, L. acidophilus, L. delbrueckii, L. brevis, L. fermentum, L. jensenii, L. plantarum, L. sake, L. curvatus and L. reuteri. Within a species, the strains exhibited highly similar patterns: the apparent molecular weights of the lytic bands were identical, with only slight variations of intensity. Moreover, each species, including phylogenetically close species such as L. sake and L. curvatus, or L. acidophilus and L. helveticus, gave a different pattern. Interestingly, the closer the species were phylogenetically, the more related were their patterns. The sensitivity of the method was checked using various quantities of L. acidophilus cells: a peptidoglycan hydrolase extract of 5 x 10(6) cells was sufficient to obtain an informative pattern, as was a single colony. Finally, the method was also successfully applied to distinguish two Carnobacterium species. In conclusion, the electrophoretic pattern of peptidoglycan hydrolases is proposed as a new tool for lactobacilli identification: it is rapid, sensitive and effective even for phylogenetically close species. Furthermore, this work provides the first evidence of the potential overall taxonomic value of bacterial peptidoglycan hydrolases.

Multilocus sequence typing of Propionibacterium freudenreichii

Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. This study investigates the molecular diversity and the population structure of this bacterium via multilocus sequence typing (MLST). Internal fragments of seven genes sequenced for 113 strains of different subspecies and origins allowed the resolution of 46 sequence types (STs) with occurrence frequencies ranging from 1 to 11. The core genome of the species harbours a low level of nucleotide polymorphism. In our data, single nucleotide polymorphisms account for only 2.28% of the concatenated sequences, and the average polymorphism rate in pairwise comparisons is 0.46%. The analyses reveal quantitatively comparable contributions of recombination and mutation in nucleotide changes at core genome loci along cell lineages. Remarkably, the STs exhibit little if any dairy biotope specialization. Phenotypic characterisation of the strains, based on their aptitude to use lactose and nitrate, shows that the two previously identified subspecies (freudenreichii and shermani) do not reflect the ancestral relationships in the P. freudenreichii population. The considerable phenotypic heterogeneity, found even at the ST level, suggests instead a history of recurrent switches between phenotypes.

Structural studies of the cell wall polysaccharides from three strains of Lactobacillus helveticus with different autolytic properties: DPC4571, BROI, and LH1

Lactobacillus helveticus is traditionally used in dairy industry as a starter or an adjunct culture for manufacture of cheese and some types of fermented milk. Its autolysis releases intracellular enzymes which is a prerequisite for optimum cheese maturation, and is known to be strain dependent. Autolysis is caused by an enzymatic hydrolysis of the cell wall peptidoglycan (PG) by endogenous peptidoglycan hydrolases (PGHs) or autolysins. Origins of differences in autolytic properties of different strains are not fully elucidated. Regulation of autolysis possibly depends on the structure of the cell wall components other than PG, particularly polysaccharides. In the present work, we screened six L. helveticus strains with different autolytic properties: DPC4571, BROI and LH1. We established, for the first time, that cell walls (CWs) of these strains contained polysaccharides, different from their CW teichoic acids. Cell wall polysaccharides of three strains were purified, and their chemical structures were established by 2D NMR spectroscopy and methylation analysis. The structures of their repeating units are presented.

Great interspecies and intraspecies diversity of dairy propionibacteria in the production of cheese aroma compounds

Flavor is an important sensory property of fermented food products, including cheese, and largely results from the production of aroma compounds by microorganisms. Propionibacterium freudenreichii is the most widely used species of dairy propionibacteria; it has been implicated in the production of a wide variety of aroma compounds through multiple metabolic pathways and is associated with the flavor of Swiss cheese. However, the ability of other dairy propionibacteria to produce aroma compounds has not been characterized. This study sought to elucidate the effect of interspecies and intraspecies diversity of dairy propionibacteria on the production of aroma compounds in a cheese context. A total of 76 strains of Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium thoenii, and Propionibacterium acidipropionici were grown for 15 days in pure culture in a rich medium derived from cheese curd. In addition, one strain each of two phylogenetically related non-dairy propionibacteria, Propionibacterium cyclohexanicum and Propionibacterium microaerophilum were included. Aroma compounds were analyzed using headspace trap-gas chromatography-mass spectrometry (GC-MS). An analysis of variance performed on GC-MS data showed that the abundance of 36 out of the 45 aroma compounds detected showed significant differences between the cultures. A principal component analysis (PCA) was performed for these 36 compounds. The first two axes of the PCA, accounting for 60% of the variability between cultures, separated P. freudenreichii strains from P. acidipropionici strains and also differentiated P. freudenreichii strains from each other. P. freudenreichii strains were associated with greater concentrations of a variety of compounds, including free fatty acids from lipolysis, ethyl esters derived from these acids, and branched-chain acids and alcohols from amino acid catabolism. P. acidipropionici strains produced less of these compounds but more sulfur-containing compounds from methionine catabolism. Meanwhile, branched-chain aldehydes and benzaldehyde were positively associated with certain strains of P. jensenii and P. thoenii. Moreover, the production of compounds with a common origin was correlated. Compound abundance varied significantly by strain, with fold changes between strains of the same species as high as in the order of 500 for a single compound. This suggests that the diversity of dairy propionibacteria can be exploited to modulate the flavor of mild cheeses.

Antifungal Microbial Agents for Food Biopreservation—A Review

Food spoilage is a major issue for the food industry, leading to food waste, substantial economic losses for manufacturers and consumers, and a negative impact on brand names. Among causes, fungal contamination can be encountered at various stages of the food chain (e.g., post-harvest, during processing or storage). Fungal development leads to food sensory defects varying from visual deterioration to noticeable odor, flavor, or texture changes but can also have negative health impacts via mycotoxin production by some molds. In order to avoid microbial spoilage and thus extend product shelf life, different treatments—including fungicides and chemical preservatives—are used. In parallel, public authorities encourage the food industry to limit the use of these chemical compounds and develop natural methods for food preservation. This is accompanied by a strong societal demand for ‘clean label’ food products, as consumers are looking for more natural, less severely processed and safer products. In this context, microbial agents corresponding to bioprotective cultures, fermentates, culture-free supernatant or purified molecules, exhibiting antifungal activities represent a growing interest as an alternative to chemical preservation. This review presents the main fungal spoilers encountered in food products, the antifungal microorganisms tested for food bioprotection, and their mechanisms of action. A focus is made in particular on the recent in situ studies and the constraints associated with the use of antifungal microbial agents for food biopreservation.

The nine peptidoglycan hydrolases genes in Lactobacillus helveticus are ubiquitous and early transcribed

Peptidoglycan hydrolases (PGHs) are bacterial enzymes that can hydrolyze the peptidoglycan in bacterial cell wall leading to autolysis. By releasing intracellular enzymes, autolysis of Lactobacillus helveticus has important applications in cheese ripening as its extent varied from strain to strain. Nine genes coding PGHs were previously annotated in the genome of the high autolytic strain L. helveticus DPC 4571. This study was conducted to evaluate the clone diversity of the nine PGHs genes within a collection of 24 L. helveticus strains, highly diverse in terms of origin, biotope and autolytic activity. Pulsed field gel electrophoresis was applied to assess the genomic diversity of the 24 strains. The presence or absence of nine PGHs genes was verified for all L. helveticus strains. Nucleotide and deduced amino acid sequence were compared for six relevant strains. Finally, gene expression was monitored by reverse transcription during growth and by zymogram for 12 strains. The nine PGHs genes are ubiquitous and transcripted early during growth. Zymograms were similar in terms of molecular size of the bands, but exhibited strain to strain variations in the number of bands revealing from 2 to 5 lytic bands per strain.