Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

Mo-HLPs: New flocculating agents identified from Moringa oleifera seeds belong to the hevein-like peptide family.

Cationic peptides found in Moringa oleifera seeds belong to different protein families and are described as the main flocculating agents of the species. In this study we report the identification and isolation of four new flocculant peptides, called Mo-HLPs 1-4, belonging to the family of hevein-like peptides, previously only known for their members' antimicrobial activity. Purification of the peptides followed two sequential membrane ultrafiltration steps and separation by reverse-phase liquid chromatography. Proteomic analyses showed that Mo-HLPs are extremely basic (pI >10) cysteine-rich molecules with molecular masses between 4.5 and 4.8 kDa and with a highly conserved chitin-binding domain. Searches in BLAST revealed high similarity of Mo-HLPs with hevein and other hevein-like peptides and 90% identity with morintides, which are members of the 8C-hevein-like subfamily found in M. oleifera leaves. Mo-HLPs microflocculation assays showed distinct coagulation/flocculation efficiencies, promoting turbidity reduction levels between 67 and 89% in synthetic turbid water. Activity variations were attributed to the substitution of some amino acids among the isoforms, which may have altered the final net charge of the molecules. The identification of Mo-HLPs represents the discovery of a new group of cationic peptides involved in the flocculation properties of M. oleifera seeds. SIGNIFICANCE: The study reveals the presence of hevein-like peptides in Moringa oleifera seeds. It is reported for the first time that members of this family have properties to act as flocculating agents of importance for water treatment processes. The identification of these peptides as well as new functional assignment broadens the horizon for speculation on new species which could act as sources of green coagulants for sustainable water treatment, and contributes to the knowledge about occurrence, distribution, molecular and active diversity of peptides belonging to the hevein-like family.

Computer aided identification of a Hevein-like antimicrobial peptide of bell pepper leaves for biotechnological use.

BACKGROUND: Antimicrobial peptides from plants present mechanisms of action that are different from those of conventional defense agents. They are under-explored but have a potential as commercial antimicrobials. Bell pepper leaves ('Magali R') are discarded after harvesting the fruit and are sources of bioactive peptides. This work reports the isolation by peptidomics tools, and the identification and partially characterization by computational tools of an antimicrobial peptide from bell pepper leaves, and evidences the usefulness of records and the in silico analysis for the study of plant peptides aiming biotechnological uses. RESULTS: Aqueous extracts from leaves were enriched in peptide by salt fractionation and ultrafiltration. An antimicrobial peptide was isolated by tandem chromatographic procedures. Mass spectrometry, automated peptide sequencing and bioinformatics tools were used alternately for identification and partial characterization of the Hevein-like peptide, named HEV-CANN. The computational tools that assisted to the identification of the peptide included BlastP, PSI-Blast, ClustalOmega, PeptideCutter, and ProtParam; conventional protein databases (DB) as Mascot, Protein-DB, GenBank-DB, RefSeq, Swiss-Prot, and UniProtKB; specific for peptides DB as Amper, APD2, CAMP, LAMPs, and PhytAMP; other tools included in ExPASy for Proteomics; The Bioactive Peptide Databases, and The Pepper Genome Database. The HEV-CANN sequence presented 40 amino acid residues, 4258.8 Da, theoretical pI-value of 8.78, and four disulfide bonds. It was stable, and it has inhibited the growth of phytopathogenic bacteria and a fungus. HEV-CANN presented a chitin-binding domain in their sequence. There was a high identity and a positive alignment of HEV-CANN sequence in various databases, but there was not a complete identity, suggesting that HEV-CANN may be produced by ribosomal synthesis, which is in accordance with its constitutive nature. CONCLUSIONS: Computational tools for proteomics and databases are not adjusted for short sequences, which hampered HEV-CANN identification. The adjustment of statistical tests in large databases for proteins is an alternative to promote the significant identification of peptides. The development of specific DB for plant antimicrobial peptides, with information about peptide sequences, functional genomic data, structural motifs and domains of molecules, functional domains, and peptide-biomolecule interactions are valuable and necessary.

Skin secretion peptides: the molecular facet of the deimatic behavior of the four-eyed frog, Physalaemus nattereri (Anura, Leptodactylidae).

RATIONALE: Amphibians can produce a large amount of bioactive peptides over the skin. In order to map the precise tissue localization of these compounds and evaluate their functions, mass spectrometry imaging (MSI) and gene expression studies were used to investigate a possible correlation between molecules involved in the antimicrobial defense mechanisms and anti-predatory behavior by Physalaemus nattereri. METHODS: Total skin secretion of P. nattereri was analyzed by classical Protein Chemistry and proteomic techniques. Intact inguinal macroglands were dissected from the rest of the skin and both tissues were analyzed by MSI and real-time polymerase chain reaction (RT-PCR) experiments. Peptides were primarily identified by de novo sequencing, automatic Edman degradation and cDNA data. RESULTS: Fifteen bradykinin (BK)-related peptides and two antimicrobial peptides were sequenced and mapped by MSI on the inguinal macrogland and the rest of P. nattereri skin. RT-PCR results revealed that BK-related peptide levels of expression were about 30,000 times higher on the inguinal macroglands than on the any other region of the skin, whilst antimicrobial peptide ions appear to be evenly distributed in both investigated regions. CONCLUSIONS: The presence of antimicrobial peptides in all investigated tissue regions is in accordance with the defensive role against microorganisms thoroughly demonstrated in the literature, whereas BK-related molecules are largely found on the inguinal macroglands suggesting an intriguing link between their noxious activities against potential predators of P. nattereri and the frog's deimatic behavior.

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

Cloning, purification, and partial characterization of Bacillus subtilis urate oxidase expressed in Escherichia coli.

Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of approximately 60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37 degrees C, respectively, and retained 90% of its activity after 72 hours of incubation at -20 degrees C and 4 degrees C.

Effect of a Bowman-Birk proteinase inhibitor from Phaseolus coccineus on Hypothenemus hampei gut proteinases in vitro.

The coffee berry borer, Hypothenemus hampei (Ferrari), is an important devastating coffee pest worldwide. Both trypsin and chymotrypsin enzyme activities from H. hampei larval midgut can be inactivated by proteinaceous enzyme-inhibitors. A serine proteinase inhibitor belonging to the Bowman-Birk class was purified from a wild accession of Phaseolus coccineus L. seeds. The inhibitor (PcBBI1) is a cysteine-rich protein that is heat-stable at alkaline pH. MALDI-TOF/MS analysis showed that PcBBI1 occurs in seeds as a monomer (8689 Da) or dimer (17,378 Da). Using in vitro inhibition assays, it was found that PcBBI1 has a high inhibitory activity against H. hampei trypsin-like enzymes, bovine pancreatic chymotrypsin, and trypsin. According to this, PcBBI1 could be a promising tool to make genetically modified coffee with resistance to coffee berry borer.