Metabolomic insights into the inhibition mechanism of methyl N-methylanthranilate: A novel quorum sensing inhibitor and antibiofilm agent against Pseudomonas aeruginosa.

The quorum sensing (QS) inhibition effect of methyl N-methylanthranilate (MMA) from Pericarpium Citri Reticulatae Chachiensis against foodborne pathogen Pseudomonas aeruginosa was reported for the first time. MMA effectively attenuated QS related virulence factors production and biofilm formation, while suppressed expression of a dozen of QS related genes. Untargeted LC-MS metabolomics revealed 108 significantly altered metabolites after MMA treatment. They indicated that MMA addition reduced the efficiency of TCA cycle and antioxidant systems, disturbed amino acid and nucleotide metabolism, increased unsaturated fatty acid and decreased peptidoglycan components, which might ultimately attenuate P. aeruginosa pathogenicity and restrain biofilm formation. Physiological characterization confirmed the compromised membrane integrity and increased intracellular oxidative stress after MMA treatment. Furthermore, metabolomics data implied that MMA inhibition on QS might exert through disrupting QS autoinducer PQS biosynthesis, which was supported by molecular docking. Our data indicated that MMA could be used as a novel QS inhibitor and anti-biofilm agent to improve food safety. It also provided new insight in the possible underlying inhibition mechanism of MMA and the response of P. aeruginosa to MMA.

Probiotic Potential and Cholesterol-Lowering Capabilities of Bacterial Strains Isolated from Pericarpium Citri Reticulatae 'Chachiensis'.

Pericarpium Citri Reticulatae 'Chachiensis' (PCR-Chachiensis), the pericarps of Citri Reticulatae Blanco cv. Chachiensis, is a food condiment and traditional medicine in southeast and eastern Asia. Its rich and various bacterial community awaits exploration. The present study is the first report on probiotic screening and characterization of bacteria from PCR-Chachiensis. Based on 64 culturable bacterial isolates, 8 strains were screened out to have great survival in the simulated gastrointestinal stressful condition, being nonhemolytic and without biogenic amine formation. They were identified by 16S rRNA gene sequencing as two Bacillus, three Lactobacillus, and three strains from Bacillales. Their probiotic properties, cholesterol-lowering potential and carbohydrate utilization capability were further investigated. Though these eight strains all displayed distinct cholesterol removal potential, Bacillus licheniformis N17-02 showed both remarkable cholesterol removal capability and presence of bile salt hydrolase gene, as well as possessing most of the desirable probiotic attributes. Thus, it could be a good probiotic candidate with hypocholesterolemic potential. Bacillus megaterium N17-12 displayed the widest carbohydrate utilization profile and the strongest antimicrobial activity. Hence, it was promising to be used as a probiotic in a host and as a fermentation starter in fermented food or feed.

In Vivo/In Vitro Properties of Novel Antioxidant Peptide from Pinctada fucata.

Due to the potential of antioxidants to scavenge free radicals in human body, it is important to be able to prepare antioxidant peptides that meet the industrial requirements for cosmetics and food. Here, we determined in vivo/in vitro activities of antioxidant peptide from P. fucata (PFAOP) prepared by bio-fermentation method. The antioxidant property test results showed the DPPH, hydroxyl, superoxide radical-scavenging, and cellular antioxidant activity. EC50 values of PFAOPs were 0.018 ± 0.005, 0.126 ± 0.008, 0.168 ± 0.005, and 0.105 ± 0.005 mg/ml, respectively, exhibiting higher antioxidant activities than glutathione (p < 0.05). Moreover, anti-proliferation and cytotoxicity activity results illustrated PFAOP has a potent anti-proliferative activity against HepG2, Caco-2, and MCF-7 carcinoma cells with no cytotoxicity. Moreover, the protocols we developed in this work demonstrated several excellent advantages in PFAOP preparation compared to enzymatic hydrolysis or chemical synthesis methods and provide a theoretical foundation for higher-value application of marine-derived functional peptides.

Molecular modification, expression and purification of new subtype antioxidant peptide from Pinctada fucata by recombinant Escherichia coli to improve antioxidant-activity.

The aim of this study was to establish a system for the efficient expression and purification of new subtype of antioxidant peptide from Pinctada fucata meat (NPFMAP), which is designed by molecular modification technology based on the sequence of purified and identified antioxidant peptide from Pinctada fucata meat (PFMAP, Gly-Ala-Gly-Leu-Pro-Gly-Lys-Arg-Glu-Arg), and to better understand the relationship between structure and antioxidant activity. Meanwhile, gene codon usage was optimized and the glutathione S-transferase (GST) tag of pGEX-6P-1 was added to facilitate expression and purification NPFMAP in Escherichia coli. The results of antioxidant activity assay in vitro showed a higher antioxidant activity in NPFMAP than that in enzymatic hydrolysis digested or chemically synthesized PFMAP. In particular, the DPPH scavenging radical activity increased by about 4.7 times after molecular modification. Structural bioanalysis indicated that new subtype antioxidant peptide had spatial conformation and good hydrophilic after modification, which was confirmed by antioxidant activity assays. Thus, the proposed method could be used to obtain NPFMAP with high antioxidant activity.

Diallyl disulfide from garlic oil inhibits Pseudomonas aeruginosa virulence factors by inactivating key quorum sensing genes.

Garlic oil can disrupt the quorum sensing (QS) pathways of the opportunistic pathogen Pseudomonas aeruginosa; however, the underlying mechanisms for this effect are unclear. Diallyl disulfide (DADS) is one of the most abundant sulfur-containing compounds in garlic oil. This study investigated the effects of DADS on the growth, virulence factor production (elastase, pyocyanin, biofilm, and swarming motility), and essential gene expression of P. aeruginosa PAO1, particularly as they apply to QS and virulence. DADS at 1.28 mg/mL did not affect P. aeruginosa PAO1 growth, although it decreased elastase and pyocyanin production, biofilm formation, and swarming motility. Each of these phenomena is regulated by the three QS systems of P. aeruginosa PAO1 (las, rhl, and pqs). Real-time q-PCR revealed that DADS down-regulated the transcription levels of several important QS genes (lasI, lasR, rhlI, rhlR, pqsA, and pqsR) in the three systems. Furthermore, the transcription levels of QS-regulated virulence genes were also down-regulated. The lasB gene, encoding LasB elastase, is co-regulated by the las, rhl, and pqs systems, and thus the down-regulation of genes across the three systems further down-regulated lasB. Additionally, phzM (encoding pyocyanin), pslB (responsible for the production of a biofilm matrix polysaccharide), and chiC (encoding chitinase) were positively activated by LasR, and a decrease in lasR transcription further down-regulated the transcription of phzM, pslB, and chiC. Hence, DADS inhibits P. aeruginosa PAO1 virulence factors by inactivating the transcription of key genes across three different QS systems.

Relationship between primary structure or spatial conformation and functional activity of antioxidant peptides from Pinctada fucata.

Antioxidant peptides from Pinctada fucata are ubiquitous in nature and can be utilized as ingredients in cosmetics or functional foods to positively regulate oxidative species in the human body against oxidation. Here, mutated peptides and a homologous series of peptides were designed and prepared, based on the original peptide sequence of an antioxidant peptide from Pinctada fucata meat (PFMAP), to better understand the structural relationships, including primary structure, spatial conformation and activity. The results showed that the antioxidant activity factors in order of importance were spatial conformation, amino acid sequence, amino acid position, amino acid composition, peptide chain length and degree of side-chain amino acid glycosylation. Moreover, this protocol had incomparable advantages in purity, speed and cost of preparing polypeptides, relative to enzymatic hydrolysis or chemosynthesis methods. This study also supplies test data information and a theoretical basis for the higher-value application of polypeptides from marine sources.

Preparation and Antioxidant Activities In Vitro of a Designed Antioxidant Peptide from Pinctada fucata by Recombinant Escherichia coli.

An antioxidant peptide derived from Pinctada fucata meat using an Alcalase2.4L enzymatic hydrolysis method (named AOP) and identified by LC-TOF-MS has promising clinical potential for generating cosmetic products that protect skin from sunshine. To date, there have been few published studies investigating the structure-activity relationship in these peptides. To prepare antioxidant peptides better and improve their stability, the design and expression of an antioxidant peptide from Pinctada fucata (named DSAOP) was studied. The peptide contains a common precursor of an expression vector containing an α-helix tandemly linked according to the BamHI restriction sites. The DNA fragments encoding DSAOP were synthesized and subcloned into the expression vector pET-30a (+), and the peptide was expressed mostly as soluble protein in recombinant Escherichia coli. Meanwhile, the DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity of DSAOP IC50 values were 0.136 ± 0.006, 0.625 ± 0.025, and 0.306 ± 0.015 mg/ml, respectively, with 2-fold higher DPPH radical scavenging activity compared with chemosynthesized AOP (p < 0.05), as well as higher superoxide radical scavenging activity compared with natural AOP (p < 0.05). This preparation method was at the international advanced level. Furthermore, pilot-scale production results showed that DSAOP was expressed successfully in fermenter cultures, which indicated that the design strategy and expression methods would be useful for obtaining substantial amounts of stable peptides at low costs. These results showed that DSAOP produced with recombinant Escherichia coli could be useful in cosmetic skin care products, health foods, and pharmaceuticals.

Diallyl Disulfide From Garlic Oil Inhibits Pseudomonas aeruginosa Quorum Sensing Systems and Corresponding Virulence Factors.

Previously, we determined that diallyl disulfide (DADS) from garlic oil can inhibit Pseudomonas aeruginosa PAO1 pathogenic factors by inactivating the transcription of key genes from three quorum sensing (QS) systems (las, rhl, and pqs) based on the effects of DADS on growth, virulence factor production (elastase, pyocyanin, biofilm, and swarming motility), and RNA transcription (real-time q-PCR). To further investigate the mechanisms underlying the inhibition of the three P. aeruginosa QS systems by DADS, high-throughput RNA and proteome sequencing techniques were used to study differences in the transcriptional and proteome expression of P. aeruginosa PAO1 following treatment with DADS. The RNA-seq and proteomic data are available via NCBI Gene Expression Omnibus database with accession number GSE118801 and ProteomeXchange with identifier PXD011144, respectively. The experimental results indicated that all key genes of the three QS systems (las, rhl, and pqs) of P. aeruginosa PAO1 as well as the virulence factors (including exoprotease LasA, elastase LasB, lectin LecA and LecB, pyocyanin biosynthesis, and biofilm formation) regulated by these three QS systems were inhibited. This is consistent with our previous studies on the physiology, biochemistry, and RNA expression of P. aeruginosa treated with DADS. Additionally, our results also indicated that bacterial motility, chemotaxis, and two-component systems were inhibited by DADS treatment. All these changes abolish the sensitivity of P. aeruginosa PAO1 to environmental stimuli and cause the cells to be in a state of passivation. Further research is needed to determine how QS systems regulate these functions. Our findings could potentially contribute to the treatment and control of P. aeruginosa infection, virulence, and pathogenicity.