De novo design of antimicrobial and antibiofilm peptides starting from desert truffle Tirmania pinoyi peptides

With the aim of discovering new routes in the research of antimicrobials, we focused on polypeptide- enriched extracts derived from edible desert truffle mushroom Tirmania pinoyi. The extracts showed an interesting activity with MIC=50 μg/mL against Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 15442. Through mass spectrometry analysis (RP-HPLC/ nESI-MS/MS) the following eight novel peptides FDVVPKTAANFRAL, AVTVGASTLADERA, FLVGGASLKPEF, VARIFAVFNDTF, HLVDEPQNLLK, LGEYGFQNALLR, FAVNGGCAKET, SREDLHPKL were detected. To characterize them online websites were used: IAMPpred, DPBAAS, Cell-PPD, ToxinPred, HemoPI, PeptideCutter and HLP. The analysis indicated that some peptides showed negative or neutral charge, hydrophobic ratio between 42% and 67%, Boman Index < 2.25 kCal/mol. According to the “APD3: Antimicrobial Peptide Calculator and Predictor” tool of the Antimicrobial Peptide Database (APD) similarities (around 30-40%) with known antimicrobial peptides (AMPs) identified in amphibians were also detected. In contrast, the predicted antimicrobial, antifungal and antibiofilm activity was not significant. In order to improve biological and physico-chemical properties, the sequences of natural peptides were modified using APD3, by replacing some hydrophilic and negative charged amino acids with hydrophobic and positive ones. The derivative sequences (GWDVVPKTWWKFRAL, KWTWGASTLAKKRA, FLRGGWSLKPKF, KWRIFWVFNKTF, HLVKRWQNLLK, KGKYRFWNALLR, FARWGGCAKRT, SRKWLHPWL) showed net positive charge between +2 and +4, hydrophobic ratio between 42% and 48%, Boman Index < 2.25 kCal/mol and high stability. Moreover, the predicted antimicrobial, antifungal and antibiofilm activity was high, without toxic or hemolytic effects. In conclusion, bioinformatic analysis has demonstrated that novel peptides discovered in T. pinoyi may be considered new platforms for the design of novel antimicrobial and antibiofilm peptides to counteract multi-drug resistant pathogens

In Vitro Cytotoxic Effect of Aqueous Extracts from Leaves and Rhizomes of the Seagrass Posidonia oceanica (L.) Delile on HepG2 Liver Cancer Cells: Focus on Autophagy and Apoptosis

Aqueous extracts from Posidonia oceanica’s green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose– response manner, with a mean half maximal inhibitory concentration (IC50) estimated at 83 and 11.5 µg of dry extract/mL, respectively. Exposure to the IC50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizomederived preparation. The underlying death-promoting mechanisms identified involved the downregulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer propertie

Removal and Reconstitution of the Carotenoid Antenna of Xanthorhodopsin

Salinixanthin, a C40-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein–lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the β-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer

The Twin-Arginine Translocation Pathway in α-Proteobacteria Is Functionally Preserved Irrespective of Genomic and Regulatory Divergence

The twin-arginine translocation (Tat) pathway exports fully folded proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Although much progress has been made in unraveling the molecular mechanism and biochemical characterization of the Tat system, little is known concerning its functionality and biological role to confer adaptive skills, symbiosis or pathogenesis in the α-proteobacteria class. A comparative genomic analysis in the α-proteobacteria class confirmed the presence of tatA, tatB, and tatC genes in almost all genomes, but significant variations in gene synteny and rearrangements were found in the order Rickettsiales with respect to the typically described operon organization. Transcription of tat genes was confirmed for Anaplasma marginale str. St. Maries and Brucella abortus 2308, two α-proteobacteria with full and partial intracellular lifestyles, respectively. The tat genes of A. marginale are scattered throughout the genome, in contrast to the more generalized operon organization. Particularly, tatA showed an approximately 20-fold increase in mRNA levels relative to tatB and tatC. We showed Tat functionality in B. abortus 2308 for the first time, and confirmed conservation of functionality in A. marginale. We present the first experimental description of the Tat system in the Anaplasmataceae and Brucellaceae families. In particular, in A. marginale Tat functionality is conserved despite operon splitting as a consequence of genome rearrangements. Further studies will be required to understand how the proper stoichiometry of the Tat protein complex and its biological role are achieved. In addition, the predicted substrates might be the evidence of role of the Tat translocation system in the transition process from a free-living to a parasitic lifestyle in these α-proteobacteria