Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH2O), Thr (-CH3CHO) and Asp (-H2O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert. Copyright (C) 2002 John Wiley Sons, Ltd.

Genetic diversity and epidemiology of antimicrobial resistant Streptococcus pneumoniae isolates

Dissertation presented to obtain a Ph.D. degree in Biology, speciality in Microbiology, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca2+ binding through EF-hand motifs and binding of Zn2+ and Cu2+ at additional sites, usually at the homodimer interfaces. Ca2+ binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.

Characterization and Biological Activities of Ocellatin Peptides from the Skin Secretion of the Frog Leptodactylus pustulatus

Eight new peptides were isolated from the skin secretion of the frog Leptodactylus pustulatus and their amino acid sequences determined by de novo sequencing and by cDNA cloning. Structural similarities between them and other antimicrobial peptides from the skin secretion of Leptodactylus genus frogs were found. Ocellatins-PT1 to -PT5 (25 amino acid residues) are amidated at the C-terminus, while ocellatins-PT6 to -PT8 (32 amino acid residues) have free carboxylates. Antimicrobial activity, hemolytic tests, and cytotoxicity against a murine fibroblast cell line were investigated. All peptides, except for ocellatin-PT2, have antimicrobial activity against at least one Gram negative strain. Ocellatin-PT8 inhibited the growth of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella choleraesuis strains with MICs in the 60−240 μM range. No significant effect was observed in human erythrocytes and in a murine fibroblast cell line after exposure to the peptides at MICs. A comparison between sequences obtained by both direct HPLC-MS de novo sequencing and cDNA cloning demonstrates the secretion of mature peptides derived from a pre-pro-peptide structure.

Exploring silk based biomaterials functionalized with antimicrobial peptides to prevent surgical site infections

Surgical site infections (SSI) often occur after invasive surgery, which is as a serious health problem, making it important to develop new biomaterials to prevent infections. Spider silk is a natural biomaterial with excellent biocompatibility, low immunogenicity and controllable biodegradability. Through recombinant DNA technology, spider silk-based materials can be bioengineered and functionalized with antimicrobial (AM) peptides 1. The aim of this study is to develop new materials by combining spider silk chimeric proteins with AM properties and silk fibroin extracted from Bombyx mori cocoons to prevent microbial infection. Here, spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6 mer and 15 mer) were fused with the AM peptides Hepcidin and Human Neutrophil peptide 1 (HNP1). The spider silk domain maintained its self-assembly features allowing the formation of beta-sheets to lock in structures without any chemical cross-linking. The AM properties of the developed chimeric proteins showed that 6 mer + HNP1 protein had a broad microbicidal activity against pathogens. The 6 mer + HNP-1 protein was then assembled with different percentages of silk fibroin into multifunctional films. In vitro cell studies with a human fibroblasts cell line (MRC5) showed nontoxic and cytocompatible behavior of the films. The positive cellular response, together with structural properties, suggests that this new fusion protein plus silk fibroin may be good candidates as multifunctional materials to prevent SSI.

Structural prediction of peptides bound to MHC class I.

An ab initio structure prediction approach adapted to the peptide-major histocompatibility complex (MHC) class I system is presented. Based on structure comparisons of a large set of peptide-MHC class I complexes, a molecular dynamics protocol is proposed using simulated annealing (SA) cycles to sample the conformational space of the peptide in its fixed MHC environment. A set of 14 peptide-human leukocyte antigen (HLA) A0201 and 27 peptide-non-HLA A0201 complexes for which X-ray structures are available is used to test the accuracy of the prediction method. For each complex, 1000 peptide conformers are obtained from the SA sampling. A graph theory clustering algorithm based on heavy atom root-mean-square deviation (RMSD) values is applied to the sampled conformers. The clusters are ranked using cluster size, mean effective or conformational free energies, with solvation free energies computed using Generalized Born MV 2 (GB-MV2) and Poisson-Boltzmann (PB) continuum models. The final conformation is chosen as the center of the best-ranked cluster. With conformational free energies, the overall prediction success is 83% using a 1.00 Angstroms crystal RMSD criterion for main-chain atoms, and 76% using a 1.50 Angstroms RMSD criterion for heavy atoms. The prediction success is even higher for the set of 14 peptide-HLA A0201 complexes: 100% of the peptides have main-chain RMSD values < or =1.00 Angstroms and 93% of the peptides have heavy atom RMSD values < or =1.50 Angstroms. This structure prediction method can be applied to complexes of natural or modified antigenic peptides in their MHC environment with the aim to perform rational structure-based optimizations of tumor vaccines.

SwissSidechain: a molecular and structural database of non-natural sidechains.

Amino acids form the building blocks of all proteins. Naturally occurring amino acids are restricted to a few tens of sidechains, even when considering post-translational modifications and rare amino acids such as selenocysteine and pyrrolysine. However, the potential chemical diversity of amino acid sidechains is nearly infinite. Exploiting this diversity by using non-natural sidechains to expand the building blocks of proteins and peptides has recently found widespread applications in biochemistry, protein engineering and drug design. Despite these applications, there is currently no unified online bioinformatics resource for non-natural sidechains. With the SwissSidechain database (http://www.swisssidechain.ch), we offer a central and curated platform about non-natural sidechains for researchers in biochemistry, medicinal chemistry, protein engineering and molecular modeling. SwissSidechain provides biophysical, structural and molecular data for hundreds of commercially available non-natural amino acid sidechains, both in l- and d-configurations. The database can be easily browsed by sidechain names, families or physico-chemical properties. We also provide plugins to seamlessly insert non-natural sidechains into peptides and proteins using molecular visualization software, as well as topologies and parameters compatible with molecular mechanics software.

Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.

The Molecular Basis for Antimicrobial Activity of Pore-Forming Cyclic Peptides

The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1–2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptides

Effect of cattle on Salmonella carriage, diversity and antimicrobial resistance in free-ranging wild boar (Sus scrofa) in northeastern Spain

Salmonella is distributed worldwide and is a pathogen of economic and public health importance. As a multi-host pathogen with a long environmental persistence, it is a suitable model for the study of wildlife-livestock interactions. In this work, we aim to explore the spill-over of Salmonella between free-ranging wild boar and livestock in a protected natural area in NE Spain and the presence of antimicrobial resistance. Salmonella prevalence, serotypes and diversity were compared between wild boars, sympatric cattle and wild boars from cattle-free areas. The effect of age, sex, cattle presence and cattle herd size on Salmonella probability of infection in wild boars was explored by means of Generalized Linear Models and a model selection based on the Akaike’s Information Criterion. Prevalence was higher in wild boars co-habiting with cattle (35.67%, CI 95% 28.19–43.70) than in wild boar from cattle-free areas (17.54%, CI 95% 8.74–29.91). Probability of a wild boar being a Salmonella carrier increased with cattle herd size but decreased with the host age. Serotypes Meleagridis, Anatum and Othmarschen were isolated concurrently from cattle and sympatric wild boars. Apart from serotypes shared with cattle, wild boars appear to have their own serotypes, which are also found in wild boars from cattle-free areas (Enteritidis, Mikawasima, 4:b:- and 35:r:z35). Serotype richness (diversity) was higher in wild boars co-habiting with cattle, but evenness was not altered by the introduction of serotypes from cattle. The finding of a S. Mbandaka strain resistant to sulfamethoxazole, streptomycin and chloramphenicol and a S. Enteritidis strain resistant to ciprofloxacin and nalidixic acid in wild boars is cause for public health concern.

Derivatives of the Antimicrobial Peptide BP100 for Expression in Plant Systems

Production of antimicrobial peptides in plants constitutes an approach for obtaining them in high amounts. However, their heterologous expression in a practical and efficient manner demands some structural requirements such as a minimum size, the incorporation of retention signals to assure their accumulation in specific tissues, and the presence of protease cleavage amino acids and of target sequences to facilitate peptide detection. Since any sequence modification may influence the biological activity, peptides that will be obtained from the expression must be screened prior to the synthesis of the genes for plant transformation. We report herein a strategy for the modification of the antimicrobial undecapeptide BP100 that allowed the identification of analogues that can be expressed in plants and exhibit optimum biological properties. We prepared 40 analogues obtained by incorporating repeated units of the antimicrobial undecapeptide, fragments of natural peptides, one or two AGPA hinges, a Gly or Ser residue at the N-terminus, and a KDEL fragment and/or the epitope tag54 at the C-terminus. Their antimicrobial, hemolytic and phytotoxic activities, and protease susceptibility were evaluated. Best sequences contained a magainin fragment linked to the antimicrobial undecapeptide through an AGPA hinge. Moreover, since the presence of a KDEL unit or of tag54 did not influence significantly the biological activity, these moieties can be introduced when designing compounds to be retained in the endoplasmic reticulum and detected using a complementary epitope. These findings may contribute to the design of peptides to be expressed in plants

Structural, EPR and Antimicrobial Studies on Some Transition Metal Complexes of Thiosemicarbazones

Department of Applied chemistry, Cochin University of Science and Technology

Diversity and antimicrobial activity of Lactic Acid Bacteria from the gut of marine fish Rastrelliger kanagurta against fish, shrimp and human pathogens

Emergence of antibiotic resistance among aquaculture pathogens has made it necessary to look into environment friendly, effective and sustainable methods such as probiotic and immunostimulants among others.. In the present study, LAB were isolated from the gut of fish species namely Rastrelliger kanagurta and analyzed for their antibacterial activity against various fish, shrimp and human pathogens. Different LAB species such as Lactobacillus plantarum, L. bulgaricus, L. brevis and L. viridiscens were encountered in the gut of R. kanagurta. Several strains showed good activity against fish, shrimp and human pathogens. LAB from the gut of such marine species may be developed as possible probiont for environment friendly health management of fresh water, estuarine and marine species currently exploited in aquaculture