Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. Mutants with deletions in two PEA transferase genes were significantly more susceptible to β-defensins, and the triple mutant was significantly more susceptible to both α- and β-defensins, but not LL-37; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface, suggesting these three genes contribute to the addition of positively charged moieties on the cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdtA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin may be more important than defensin resistance to H. ducreyi pathogenesis.

Made available in DSpace on 2014-06-11T19:23:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-18Bitstream added on 2014-06-13T18:20:01Z : No. of bitstreams: 1 badial_pr_me_botfmvz.pdf: 583023 bytes, checksum: 32f4c9e3526cb647c9f301ae256b559c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A hepcidina é uma proteína que faz parte do sistema imune inato e desempenha um papel fundamental na regulação da homeostase do ferro. Este peptídeo foi previamente caracterizado em várias espécies, entretanto, até agora, não em ovinos. O objetivo deste estudo foi de determinar a sequência do RNAm codificante da hepcidina, bem como caracterizar e realizar a análise da expressão gênica deste peptídeo em diferentes tecidos de ovinos saudáveis. A região codificante da hepcidina consiste em 249 pares de base, as quais codificam um peptídeo contendo 82 aminoácidos. Este precursor, da forma bioativa da hepcidina, apresenta maior homologia com as sequências de Bos taurus e Bubalus bubalis. A hepcidina foi expressa predominantemente no fígado dos ovinos, contudo foram observados altos níveis de expressão no abomaso e baixos níveis nos outros tecidos. Estes resultados ampliam o conhecimento comparativo deste peptídeo, mostrando a relação da hepcidina ovina com a de outras espécies de mamíferos e será útil para estudos futuros sobre o metabolismo de ferro e do processo inflamatório nos ovinos. / Hepcidin is part of the innate immune system, and plays a central role in the regulation of iron homeostasis. This peptide has been previously characterized in several species but not in sheep until now. The aim of this study was to sequence, characterize and perform hepcidin gene expression analysis in different tissues of healthy sheep. The resulting ORF consisted of 249 bp predicted to encode an 82 amino acids peptide. The deduced precursor was mostly homologous to Bos taurus and Bubalus bubalis. Hepcidin was predominantly expressed in liver, although high expression was present in abomasum and lower level expression occurred in other tissues. These findings extend our comparative knowledge of this peptide, showing the relationship between sheep hepcidin and other mammalian hepcidins and might be helpful for additional studies on iron metabolism and inflammatory process in sheep.

Despite many medical advances, antibiotic resistant bacteria increasingly plague the modern world, necessitating discovery of new antibiotics. One area of nature that can provide inspiration for antibiotics is antimicrobial peptides. Many of these peptides exist in nature, with some classes that have not been studied or characterized well. One such class is the defensin-like peptides generated by the plant Medicago truncatula as part of their symbiotic relationship with Sinorhizobium meliloti. Nodule-specific Cysteine Rich (NCR) peptides are defined by the presence of multiple cysteines, and regulate the growth of S. meliloti within plant cells. While some of these NCR peptides have been shown to have antimicrobial properties, hundreds of peptides remain uncharacterized. We have developed an assay for further characterization of these peptides in E. coli. Of the seven peptides that have been tested using this assay, three have exhibited definitive antimicrobial properties against both E. coli and S. meliloti. Additionally, we have developed a system for discovering novel antimicrobial peptides. This platform, called PepSeq, uses the expression of random peptides in E. coli combined with deep sequencing to detect antimicrobial activity. This technology is capable of screening through millions of peptide molecules simultaneously. Using this platform, we have discovered and confirmed six novel antimicrobial peptides, with hundreds of additional predicted antimicrobial peptides. In addition to the peptides we have analyzed using PepSeq, additional peptide scaffolds could be used to discover more potent antimicrobial peptides.

No description available.

Humans are continuously exposed to mixtures of environmental pollutants. Polycyclic aromatic hydrocarbons (PAHs), such as 2-naphthol, and heavy metals, such as lead, are some of these pollutants. Results from epidemiological studies show associations between exposure to 2-naphthol, exposure to lead, and obesity. However, the individual and combined effects of 2-naphthol and lead on fat cell development (adipogenesis) have not been directly characterized in a biological system. In this study, we evaluated the effects of 2-naphthol and/or lead on adipogenesis using mouse 3T3-L1 cells. Cells were exposed to different doses of 2-naphthol and/or lead. Induced terminal differentiation was evaluated by cell morphology, lipid production, and mRNA expression of marker genes characteristic of either early adipocyte differentiation: CCAAT-enhancer-binding protein β (C/EBPβ), insulin receptor substrate 2 (IRS2), and sterol responsive element binding protein 1 c (SREBP1c); or terminal differentiation: C/EBPα, peroxisome proliferator-activated receptor-γ (PPARγ), and fatty acid binding protein 4 (aP2). Production of antimicrobial peptide cathelicidin (Camp), which is produced by differentiating adipocytes and modulates inflammation and immunity, was also evaluated. Cell morphology changes and increased lipid accumulation indicated that, individually, 2-naphthol and lead induced 3T3-L1 differentiation; however, the highest dose of lead (10 μM) showed the lowest induction level. During terminal differentiation, 2-naphthol and low doses of lead increased C/EBPα, PPARγ, and aP2 expression, whereas 10 μM lead suppressed PPARγ and aP2. During early differentiation, 2-naphthol stimulated C/EBPβ, IRS2, and SREBP1c expression, while lead upregulated C/EBPα and aP2. The 2-naphthol/10 μM lead mixture induced a counterbalancing effect on 3T3-L1 adipogenesis, where 10 μM lead suppressed 2-naphthol-induced adipogenesis. Moreover, 2-naphthol elevated Camp expression in a dose-dependent manner, whereas lead slightly increased Camp at lower doses but suppressed it at 10 μM. The 2-naphthol/10 μM lead mixture showed no effect on Camp expression. In conclusion, 2-naphthol and low lead doses accelerate adipocyte differentiation and Camp production in 3T3-L1 cells; however, high doses of lead attenuate the induction. This effect of lead at high dose counterbalances the upregulation of adipocyte differentiation and Camp production by 2-naphthol. Together, these findings indicate that 2-naphthol and lead play potential roles in the development of inflammation and obesity.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

Biological membranes are quasi two-dimensional self-assembled structure, primarily serving as a barrier to the leakage of cell’s contents. The main constituents of biological membrane are various amphiphilic lipids that form bilayers in an aqueous environment. These lipids carry acidic and/or basic functional groups that ionize in water, giving some of them a net electrical charge. Such a lipid molecule, when integrated into the membrane, experiences electrostatic forces from all other charged objects around it, including ions, surrounding lipids, and other molecules such as cationic peptides. The electrostatic interaction can profoundly influence the membrane, to which many phenomena with physiological significance as well as biophysical interest can be ascribed. In this thesis, we concentrate on investigating the electrostatic properties of lipid membranes. First, we study how the electrostatic interaction affects their preferred structure. To this end, we adopt a coarse-grain model that preserves the dominant characteristics of the lipids, in which the electrostatic interaction is treated within the “renormalized” Debye-H¨uckel theory. In particular, we calculate the spontaneous curvature of a phospholipid monolayer, along with other associated quantities. Our results suggest that such divalent ions as Mg2+ can stabilize HII phases of lipids (inverted hexagonal phases), which would otherwise form lamellar phases. Second,we investigate the competitive binding of ions and cationic peptides onto a monolayer of lipopolysaccharide (LPS) molecules, a class of highly charged bio-molecules found in the outer leaflet of the outer membranes of gram-negative (G-) bacteria. Cationic anti-microbial peptides (AMPs) can selectively kill bacteria, and it is suggested that they destabilize the LPS layer, easing their permeation across it, a process of great physiological and clinical interest. To this end, we model the LPS layer as a collection of charged “binding sites”, based on which we study the binding of cations (monovalent and divalent) and cationic peptides onto the layer. Our calculations suggest that the peptides can compete with divalent ions on the binding to the layer. It has been empirically known that since the stability of an LPS layer relies greatly on the bridging of divalent ions, the substitution of these ions by the peptides significantly compromises its stability. Our results offer a quantitative basis for this observation, thus providing a possible mechanism of an important step in the action of AMPs against G- bacteria.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

No description available.

Biological membranes are quasi two-dimensional self-assembled structure, primarily serving as a barrier to the leakage of cell’s contents. The main constituents of biological membrane are various amphiphilic lipids that form bilayers in an aqueous environment. These lipids carry acidic and/or basic functional groups that ionize in water, giving some of them a net electrical charge. Such a lipid molecule, when integrated into the membrane, experiences electrostatic forces from all other charged objects around it, including ions, surrounding lipids, and other molecules such as cationic peptides. The electrostatic interaction can profoundly influence the membrane, to which many phenomena with physiological significance as well as biophysical interest can be ascribed. In this thesis, we concentrate on investigating the electrostatic properties of lipid membranes. First, we study how the electrostatic interaction affects their preferred structure. To this end, we adopt a coarse-grain model that preserves the dominant characteristics of the lipids, in which the electrostatic interaction is treated within the “renormalized” Debye-H¨uckel theory. In particular, we calculate the spontaneous curvature of a phospholipid monolayer, along with other associated quantities. Our results suggest that such divalent ions as Mg2+ can stabilize HII phases of lipids (inverted hexagonal phases), which would otherwise form lamellar phases. Second,we investigate the competitive binding of ions and cationic peptides onto a monolayer of lipopolysaccharide (LPS) molecules, a class of highly charged bio-molecules found in the outer leaflet of the outer membranes of gram-negative (G-) bacteria. Cationic anti-microbial peptides (AMPs) can selectively kill bacteria, and it is suggested that they destabilize the LPS layer, easing their permeation across it, a process of great physiological and clinical interest. To this end, we model the LPS layer as a collection of charged “binding sites”, based on which we study the binding of cations (monovalent and divalent) and cationic peptides onto the layer. Our calculations suggest that the peptides can compete with divalent ions on the binding to the layer. It has been empirically known that since the stability of an LPS layer relies greatly on the bridging of divalent ions, the substitution of these ions by the peptides significantly compromises its stability. Our results offer a quantitative basis for this observation, thus providing a possible mechanism of an important step in the action of AMPs against G- bacteria.