Characterisation of EFV12 a bio-active small peptide produced by the human intestinal isolate Lactobacillus gasseri SF1109.

EFV12 is a small bioactive peptide produced by Lactobacillus gasseri SF1109, a human intestinal isolate with probiotic features. In this study, EFV12 antimicrobial and anti-inflammatory properties are characterised. In particular, we propose a possible mechanism of action for EFV12 involving bacterial membranes targeting. Moreover, we show that this small peptide is able to bind lipopolysaccharides (LPS) and to counteract its inflammatory insult preventing LPS action on Toll-like receptor 4, thus interfering with extracellular signal-regulated kinase, p38 and Jun N-terminal kinase, mitogen-activated protein kinases signalling pathways. Altogether these observations suggest that the bioactive peptide EFV12 is a good candidate to promote L. gasseri induced gut homeostasis and counteracting intestinal pathogens.

A new cryptic host defense peptide identified in human 11-hydroxysteroid dehydrogenase-1 ß-like: from in silico identification to experimental evidence.

BACKGROUND: Host defence peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 ß-like protein. METHODS: Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. RESULTS: GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is not toxic towards human and murine cell lines and triggers a significant innate immune response by attenuating expression levels of pro-inflammatory interleukins and release of nitric oxide in LPS induced macrophages. CONCLUSION: Human GVF27 may offer significant advantages as leads for the design of human-specific therapeutics. GENERAL SIGNIFICANCE: Human cryptic host defence peptides are naturally no immunogenic and for this they are a real alternative for solving the lack of effective antibiotics to control bacterial infections.

Identification of a new small bioactive peptide from Lactobacillus gasseri supernatant.

Ten lactic acid bacteria (LAB) strains, previously isolated from human ileal biopsy of healthy volunteers, were screened for production and secretion of molecules having anti-bacterial and anti-biofilm activities. Because many recent reports indicate that LAB secreted molecules may exert immune-modulatory action, we also tested the effect on human intestinal HCT116 cells challenged with bacterial lipopolysaccharides. One of the Lactobacillus gasseri strains, SF1109, strongly inhibited: (1) Pseudomonas aeruginosa growth; (2) Escherichia coli biofilm production; (3) LPS induction of P-ERK1/2 in HCT116 cells, and was selected for further characterisation of the secreted active molecule. Cell-free supernatant of the L. gasseri SF1109 was analysed and one 1.3 kDa peptide has been characterised. Eight out twelve amino acids of this peptide were identified allowing the synthesis of an octa-peptide which still presented the mentioned activities.

De novo sequencing and assembly of the whole genome of Novosphingobium sp. strain PP1Y.

Novosphingobium sp. strain PP1Y is a marine bacterium specifically adapted to use fuels as an energy source. We sequenced and assembled its entire genome using the Roche 454 genome sequencer system, which led to the identification of two plasmids and one megaplasmid, besides a 3.9-Mb circular chromosome.

Contribution of chain termini to the conformational stability and biological activity of onconase.

Onconase, a member of the RNase A superfamily, is a potent antitumor agent which is undergoing phase III clinical trials as an antitumor drug. We have recently shown that onconase is an unusually stable protein. Furthermore, the protein is resistant to the action of proteases, which could influence its use as a drug, prolonging its biological life, and leading to its renal toxicity. Our investigation focused on the contribution of chain termini to onconase conformational stability and biological activities. We used differential scanning calorimetry, isothermal unfolding experiments, limited proteolysis, and catalytic and antitumor activity determinations to investigate the effect of the elimination of the two blocks at the chain termini, the N-terminal cyclized glutamine and the C-terminal disulfide bridge between the terminal Cys104 and Cys87. The determination of the thermodynamic parameters of the protein led to the conclusion that the two blocks at onconase chain termini are responsible for the unusual stability of the protein. Moreover, the reduced stability of the onconase mutants does not influence greatly their catalytic and antitumor activities. Thus, our data would suggest that an onconase-based drug, with a decreased toxicity, could be obtained through the use of less stable onconase variants.

Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1.

A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building.

Onconase: an unusually stable protein.

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity.

Effective expression and purification of recombinant onconase, an antitumor protein.

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials. We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields. The recombinant protein has biological and catalytic properties identical to those of the natural enzyme.

Protein engineering of ribonucleases.

Natural bovine seminal RNase possesses a potent antitumor action. We have mutagenized monomeric bovine pancreatic RNase A, devoid of any cytotoxic action, to insert residues present at corresponding positions in the subunit of dimeric, antitumor, seminal RNase. Like naturally dimeric seminal RNase, the mutant dimeric RNases display selective toxicity for malignant cells, which is absent in the monomeric mutants.