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Propionibacterium freudenreichii MJ2-derived extracellular vesicles LC-MS/MS

ABSTRACT: PFEVs inhibit osteoclast differentiation by interfering RANK-RANKL binding, thereby decreasing expression of gene related to osteoclast differentiation and improve CIA, probably through inhibiting inflammation and osteoclastogenesis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Propionibacterium Freudenreichii Subsp. Freudenreichii

TISSUE(S): Cell Suspension Culture

SUBMITTER: Heeeun Woo

LAB HEAD: Young-Hee Lim

PROVIDER: PXD051291 | Pride | 2024-11-04

REPOSITORIES: Pride

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			Action	DRS
	KRU_exosome_01.raw	Raw
	KRU_exosome_uniprot_Anno_1.msf	Msf
	checksum.txt	Txt

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Propionibacterium freudenreichii MJ2 inhibits RANKL-induced osteoclast differentiation and ameliorates rheumatoid arthritis in collagen-induced arthritis model

Project description:Osteoclast differentiation is crucial for bone absorption and osteoclast is involved in bone destruction in rheumatoid arthritis. The aim of this study was to investigate the inhibitory effect of MJ2 on osteoclast differentiation and to elucidate its mechanism. Murine macrophage cell line, Raw 264.7 cells and collagen-induced arthritis mouse model were used for in vitro and in vivo study, respectively. MJ2-treated cells significantly inhibited osteoclast differentiation and decreased arthritic score. Surface proteins (SP) extracted from MJ2 also showed inhibitory effect on osteoclast differentiation by upregulating lipocalin 2 (lcn2) expression. Specifically, heat shock protein 60 (hsp60) in SP was revealed as an active component of MJ2. Hsp60 inhibited binding of receptor activator of nuclear factor-κB ligand (RANKL) to receptor activator of nuclear factor κB (RANK). In conclusion, MJ2 inhibited osteoclast formation and differentiation through lcn2 and RANKL-binding property and the effective component of MJ2 might be hsp60 present in surface layer.

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Emmental Cheese Environment Enhances Propionibacterium freudenreichii Stress Tolerance.

Project description:Dairy propionibacteria are actinomycetales found in various fermented food products. The main species, Propionibacterium freudenreichii, is generally recognized as safe and used both as probiotic and as cheese starter. Its probiotic efficacy tightly depends on its tolerance towards digestive stresses, which can be largely modulated by the ingested delivery vehicle. Indeed, tolerance of this bacterium is enhanced when it is consumed within a fermented dairy product, compared to a dried probiotic preparation. We investigated both stress tolerance and protein neosynthesis upon growth in i) chemically defined or ii) aqueous phase of Emmental cheeses. Although the same final population level was reached in both media, a slower growth and an enhanced survival of CIRM BIA 1 strain of P. freudenreichii subsp. shermanii was observed in Emmental juice, compared to chemically defined medium. This was accompanied by differences in substrates used and products released as well as overexpression of various early stress adaptation proteins in Emmental juice, compared to chemically defined medium, implied in protein folding, in aspartate catabolism, in biosynthesis of valine, leucine and isoleucine, in pyruvate metabolism in citrate cycle, in the propionate metabolism, as well as in oxidoreductases. All these changes led to a higher digestive stress tolerance after growth in Emmental juice. Mechanisms of stress adaptation were induced in this environment, in accordance with enhanced survival. This opens perspectives for the use of hard and semi-hard cheeses as delivery vehicle for probiotics with enhanced efficacy.

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Project description:Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.

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Propionibacterium freudenreichii transcriptomic response during cold storage

Project description:Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It produces flavor compounds over the whole ripening period. During cheese ripening, P. freudenreichii is exposed to a temperature downshift, especially when cheeses are transferred from warm temperature (about 24°C) to cold temperature (about 4°C). The aim of this study was to investigate the adaptation of P. freudenreichii at cold temperature by means of the first global gene expression profile for this species. The temporal transcriptomic response of P. freudenreichii was analyzed at five times of growth, during growth at 30°C then for 9 days at 4°C, in the constant presence of lactate as the main carbon source. P. freudenreichii response was also investigated by RT-qPCR for 30 genes, by proteomics and metabolomics (main metabolites quantified in culture supernatant). Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from warm to cold temperature (P < 0.05 and |fold change| > 1). Most of the down-expressed genes were involved in cell machinery (cell division, protein turnover, translation, transcription and DNA replication). During incubation at cold temperature, P. freudenreichii accumulated carbon supplies by up-regulating genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis and in glycogen synthesis. Interestingly, some genes involved in the formation of important flavor compounds of cheese, coding for an extracellular lipolytic esterases and enzymes of the pathways of formation of branched-chain compounds, were not significantly affected by cold. In conclusion, P. freudenreichii is metabolically active at cold temperature and induces pathways to maintain its long-term viability, which could explain its contribution to cheese ripening even at low temperature.

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