Victoria Chuat

Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties

UNLABELLED Propionibacterium freudenreichii is a beneficial bacterium used in the food industry as a vitamin producer, as a bio-preservative, as a cheese ripening starter and as a probiotic. It is known to adhere to intestinal epithelial cells and mucus and to modulate important functions of the gut mucosa, including cell proliferation and immune response. Adhesion of probiotics and cross-talk with the host rely on the presence of key surface proteins, still poorly identified. Identification of the determinants of adhesion and of immunomodulation by P. freudenreichii remains a bottleneck in the elucidation of its probiotic properties. In this report, three complementary proteomic methods are used to identify surface-exposed proteins in a strain, previously selected for its probiotic properties. The role of these proteins in the reported immunomodulatory properties of P. freudenreichii is evidenced. This work constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context. BIOLOGICAL SIGNIFICANCE Dairy propionibacteria, mainly the species Propionibacterium freudenreichii, are consumed in high amounts within Swiss type cheeses. These peculiar bacteria are considered both as dairy starters and as probiotics. Their consumption modulates the gut microbiota, which makes them both probiotic and prebiotic. Promising immunomodulatory properties have been identified in these bacteria, in vitro, in animals and in humans. However, the mechanisms responsible for such anti-inflammatory properties are still unknown. In this work, we identify surface proteins involved in adhesion and immunostimulation by P. freudenreichii. This opens new perspectives for its utilization in new functional fermented food products, in clinical trials, and in understanding modulation of gut inflammation by products containing propionibacteria.

Lactic Acid Bacteria Isolated from Bovine Mammary Microbiota: Potential Allies against Bovine Mastitis.

Bovine mastitis is a costly disease in dairy cattle worldwide. As of yet, the control of bovine mastitis is mostly based on prevention by thorough hygienic procedures during milking. Additional strategies include vaccination and utilization of antibiotics. Despite these measures, mastitis is not fully under control, thus prompting the need for alternative strategies. The goal of this study was to isolate autochthonous lactic acid bacteria (LAB) from bovine mammary microbiota that exhibit beneficial properties that could be used for mastitis prevention and/or treatment. Sampling of the teat canal led to the isolation of 165 isolates, among which a selection of ten non-redundant LAB strains belonging to the genera Lactobacillus and Lactococcus were further characterized with regard to several properties: surface properties (hydrophobicity, autoaggregation); inhibition potential of three main mastitis pathogens, Staphylococcus aureus, Escherichia coli and Streptococcus uberis; colonization capacities of bovine mammary epithelial cells (bMEC); and immunomodulation properties. Three strains, Lactobacillus brevis 1595 and 1597 and Lactobacillus plantarum 1610, showed high colonization capacities and a medium surface hydrophobicity. These strains are good candidates to compete with pathogens for mammary gland colonization. Moreover, nine strains exhibited anti-inflammatory properties, as illustrated by the lower IL-8 secretion by E. coli-stimulated bMEC in the presence of these LAB. Full genome sequencing of five candidate strains allowed to check for undesirable genetic elements such as antibiotic resistance genes and to identify potential bacterial determinants involved in the beneficial properties. This large screening of beneficial properties while checking for undesirable genetic markers allowed the selection of promising candidate LAB strains from bovine mammary microbiota for the prevention and/or treatment of bovine mastitis.

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.

The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

ABSTRACT Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.

Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese.

SCOPE Inflammatory bowel disease (IBD) constitutes a growing public health concern in western countries. Bacteria with anti-inflammatory properties are lacking in the dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti-inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters such as Propionibacterium freudenreichii and Lactobacillus delbrueckii (Ld). We thus investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients. METHODS AND RESULTS We developed a single-strain Ld-fermented milk and a two-strain P. freudenreichii and Ld-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties. Consumption of these experimental fermented dairy products protected mice against trinitrobenzenesulfonic acid induced colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection. CONCLUSION This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives for the reverse engineering development of anti-inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients.

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.

Identification of proteins involved in the anti-inflammatory properties of Propionibacterium freudenreichii by means of a multi-strain study

Propionibacterium freudenreichii, a dairy starter, can reach a population of almost 109 propionibacteria per gram in Swiss-type cheese at the time of consumption. Also consumed as a probiotic, it displays strain-dependent anti-inflammatory properties mediated by surface proteins that induce IL-10 in leukocytes. We selected 23 strains with varied anti-inflammatory potentials in order to identify the protein(s) involved. After comparative genomic analysis, 12 of these strains were further analysed by surface proteomics, eight of them being further submitted to transcriptomics. The omics data were then correlated to the anti-inflammatory potential evaluated by IL-10 induction. This comparative omics strategy highlighted candidate genes that were further subjected to gene-inactivation validation. This validation confirmed the contribution of surface proteins, including SlpB and SlpE, two proteins with SLH domains known to mediate non-covalent anchorage to the cell-wall. Interestingly, HsdM3, predicted as cytoplasmic and involved in DNA modification, was shown to contribute to anti-inflammatory activity. Finally, we demonstrated that a single protein cannot explain the anti-inflammatory properties of a strain. These properties therefore result from different combinations of surface and cytoplasmic proteins, depending on the strain. Our enhanced understanding of the molecular bases for immunomodulation will enable the relevant screening for bacterial resources with anti-inflammatory properties.

Biodiversity of dairy Propionibacterium isolated from dairy farms in Minas Gerais, Brazil

Dairy propionibacteria are used as ripening cultures for the production of Swiss-type cheeses, and some strains have potential for use as probiotics. This study investigated the biodiversity of wild dairy Propionibacteria isolates in dairy farms that produce Swiss-type cheeses in Minas Gerais State, Brazil. RAPD and PFGE were used for molecular typing of strains and MLST was applied for phylogenetic analysis of strains of Propionibacterium freudenreichii. The results showed considerable genetic diversity of the wild dairy propionibacteria, since three of the main species were observed to be randomly distributed among the samples collected from different farms in different biotopes (raw milk, sillage, soil and pasture). Isolates from different farms showed distinct genetic profiles, suggesting that each location represented a specific niche. Furthermore, the STs identified for the strains of P. freudenreichii by MLST were not related to any specific origin. The environment of dairy farms and milk production proved to be a reservoir for Propionibacterium strains, which are important for future use as possible starter cultures or probiotics, as well as in the study of prevention of cheese defects.

New insights about phenotypic heterogeneity within Propionibacterium freudenreichii argue against its division into subspecies.

Propionibacterium freudenreichii is widely used in Swiss-type cheese manufacture, where it contributes to flavour and eye development. It is currently divided into two subspecies, according to the phenotype for lactose fermentation and nitrate reduction (lac+/nit− and lac−/nit+ for P. freudenreichii subsp. shermanii and subsp. freudenreichii, respectively). However, the existence of unclassifiable strains (lac+/nit+ and lac−/nit−) has also been reported. The aim of this study was to revisit the relevance of the subdivision of P. freudenreichii into subspecies, by confirming the existence of unclassifiable strains. Relevant conditions to test the ability of P. freudenreichii for lactose fermentation and nitrate reduction were first determined, by using 10 sequenced strains, in which the presence or absence of the lactose and nitrate genomic islands were known. We also determined whether the subdivision based on lac/nit phenotype was related to other phenotypic properties of interest in cheese manufacture, in this case, the production of aroma compounds, analysed by gas chromatography-mass spectrometry, for a total of 28 strains. The results showed that a too short incubation time can lead to false negative for lactose fermentation and nitrate reduction. They confirmed the existence of four lac/nit phenotypes instead of the two expected, thus leading to 13 unclassifiable strains out of the 28 characterized (7 lac+/nit+ and 6 lac−/nit−). The production of the 15 aroma compounds detected in all cultures varied more within a lac/nit phenotype (up to 20 times) than between them. Taken together, these results demonstrate that the division of P. freudenreichii into two subspecies does not appear to be relevant.

Permanent draft genome sequence of the probiotic strain Propionibacterium freudenreichii CIRM-BIA 129 (ITG P20)

Propionibacterium freudenreichii belongs to the class Actinobacteria (Gram positive with a high GC content). This “Generally Recognized As Safe” (GRAS) species is traditionally used as (i) a starter for Swiss-type cheeses where it is responsible for holes and aroma production, (ii) a vitamin B12 and propionic acid producer in white biotechnologies, and (iii) a probiotic for use in humans and animals because of its bifidogenic and anti-inflammatory properties. Until now, only strain CIRM-BIA1T had been sequenced, annotated and become publicly available. Strain CIRM-BIA129 (commercially available as ITG P20) has considerable anti-inflammatory potential. Its gene content was compared to that of CIRM-BIA1 T. This strain contains 2384 genes including 1 ribosomal operon, 45 tRNA and 30 pseudogenes.