In Vitro Activities of Synthetic Host Defense Propeptides - Research Paper Example

Host defence peptides (HDPs) are an integral part of the innate immune response in mammalian immunity. This is because HDPs play diverse and complex roles in immune response from mediating the killing of invading microorganisms to being potent antimicrobial peptides at modest concentration [1]. In addition, mammalian HDPs also play a number of immunomodulatory functions such as altering host gene expression, inhibiting lipopolysaccharides induced proinflammatory cytokines production a well as promoting faster wound healing. Host defence peptides have been shown to be vital in prevention and clearance of infection [1]. The potential of HDPs as multifunctional effectors of innate immunity in higher organism provides the impetus and motivation for the progress in their research. The research paper being summarized in this report is In vitro activities of synthetic host defense propeptides processed by neutrophil elastase against cystic fibrosis pathogens authored by Stephen Desgranges, Florie Le Prieult, Alan Daly’ Jeniffer Lydon, Marian Brennan, Dilip K. Rai, Anusha P. Subasinghage, Chandralal M. Hewage, Sally-Ann Cryan, Catherine Greene, Noel G. McElvaney, Timothy P. Symth and Marc Devocelle. This paper was published in the journal Antimicrobial agents and chemotherapy under the American society for microbiology on May 2011. The journal has an impact factor of 4.672, a respectable impact factor for any reputable journal. Publishing of the work was significant to shed light on light on activities of synthetic host defense propeptides firsthand and hinder other person/groups doing so first. The Desgranges et al have undertaken a study where they have reported the in vitro activities of synthetic host defense propeptides against cystic pathogens namely Staphylococcus aureus and Pseudomonas aeruginosa­ [2]. This paper focuses on the effect of modifying the propeptide by reducing the net change on the antimicrobial and haemolytic activity. The aim of this work by Desgranges et al was to utilize the abnormal amounts of neutrophil elastase present in cystic fibrosis to process several synthetic prodrugs against common pathogens in cystic fibrosis, P. aeruginosa and S. aureus [2]. Peptides are present in many living systems and are utilized by the innate immune systems to combat invading pathogens. Therefore this study aims at constructing a propeptide that will be able to evade the host immune system and also be a substrate for the all abundant neutrophil elastase present in cystic fibrosis lung. Since peptides have been shown to possess antibacterial activity against bacteria where they interfere with the cell membrane; the stud aims at utilizing neutrophil elastase processing activity to deploy a peptide with antibacterial activity against two representative of cystic fibrosis lung bacteria milieu, Staphylococcus aureus and Pseudomonas aeruginosa. Cystic fibrosis is a lethal hereditary disease which affects the liver, pancreas and intestinal tract. Mortality and morbidity from the disease occurs in the lung disease [3]. Early years of the disease are presented with production of thick mucus and chronic airways infection which may lead to progressive compromise of lung function and certainly death. Lung complications which may occur in cystic fibrosis include haemoptysis, pneumothorax and pulmonary hypertension. The disease is caused by mutations which occur in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [4]. Due to mutations in this gene, coded CFTR protein has abnormalities. However the mechanism of disease causation by this abnormal CFTR protein is not well established [5]. Characteristics of cystic fibrosis are intense neutrophil-dominated inflammation where neutrophils in CF lung account for 70% of the epithelial lining fluid (ELF) inflammatory cells [5]. Pathogenesis in the lungs is mainly due to the ineffectiveness of neutrophils to clear bacteria. Neutrophil proteases and oxidants overwhelm the antiprotease activity in the lung’s epithelial surface. A major protease present in neutrophils in cystic fibrotic lung is neutrophil elastase which interferes in ciliary beat frequency [6]. This enzyme also deranges the mucus glycoprotein secretion [7]. Host defence peptides for instance defensins form part of the central innate immune elements. Instantaneous response by the elements of the innate immune response mechanism figs off the vast majority of threats before the adaptive immune response is activated. This response is made up of pattern recognition receptors (PRRs), signaling via transcriptional factors and the production of effector molecules such as HDPs. These HDPs exhibit chemokine-like activities whereas others possess antimicrobial activity. Host defence peptides mode of action ; Shai-Matsuzaki-Huang model (Linde 102) [8] Key; [A]: attachment of HDPs to microbial membrane [B]: pore formation [C]: cell death Antimicrobial activity of HDPs is mediated via pore formation in the surface membrane of pathogens orchestrating a leakage and which leads to pathogen demise [10]. Preparation of synthetic propeptides was achieve by modifying an HDP with an anionic profragment, L-P18. This is an α-helical, salt-resistant, cecropin A-magainin 2 hybrid sequence. The solid-phase synthesis was used to assemble the peptides with reversed-phase HPLC being used to separate them. In solid-phase synthesis, N-a-amino acids are incorporated into a peptide of the desired sequence while one end of the sequence remains attached on a solid support matrix. Peptide synthesis is done in a stepwise method where soluble reagents can be removed from the peptide-solid support matrix by filtration and washed away after every coupling step. Solid matrix is usually a synthetic polymer bearing -OH groups. HPLC separation technique utilizes columns and pumps to separate samples such as peptides with a high degree of resolution. In reverse HPLC the peptide was separated based on its hydrophobic interactions in the column and elution was in accordance with decreasing ionic strength. Column is composed of hydrocarbon alkane chains which are linked to the silica covalently. Characterization of the assembled peptides was by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. MALDI-TOF provides a powerful tool for fast and accurate determination of the peptides molar masses and any additives or impurities present during the synthesis process. This method is based on an ultraviolet absorbing matrix that is mixed with the polymer at molecular level in appropriate solvent. Solvent hinders the aggregation of the polymer and under vacuum conditions the solvent is removed leaving co-crystallized polymer molecules homogeneously dispersed within matrix. In this synthesis six propeptides were generated. Antimicrobial activities of the generated propeptides were assessed for two representatives of cystic fibrosis pathogens, P. aeruginosa and S. aureus by the broth microdilution method. Susceptibility testing was done in purified human neutrophil elastase at concentrations of 0.075 µg/ml and 0.15µg/ml to assess the peptides reactivation. Toxicity studies of the propeptides of the proforms of D-P18 were performed via a hemolytic assay. 1H nuclear magnetic resonance (NMR) was used to ascertain whether activity differential between the propeptides D-P18 and Ac-E4-A3-D-P18, was as a result of conformational. In the synthesis of the six propeptides the present sequence (KWKLFKKIPKFLHLAKKF-NH2) was elongated at the N terminus by a trialanine linker which is an NE substrate [10]. In order to mask the net charge determinant of mature peptide antimicrobial activity, 2-7 glutamic acids were added. If there were more than four glutamates added, a β-alanine spacer was inserted between 4th and 5th glutamates. The purpose of the spacer was to minimize interchain association [11]. An antimicrobial activity of the L-P18 MICs was found to be 32µM and 64µM for P. aeruginosa and S. aureus respectively. However the MICs for the other propeptides was fond to be high than 128µM. in respect to the stereochemistry of constitutive amino acids, activities of α-helical membrane-active peptides have been claimed to be unaffected by their stereochemistry[12]. Assembled D-P18 an enantiomer of L-P18 with an extension bearing the trialanine linker and an N-terminally acetylated tetraglutamate motif was found to have lower MICs than D-P18. This enantiomer may escape the cell proteolytic degradation due to its stereochemistry. Susceptibility testing of the assembled D-P18 done in human neutrophil elastase at concentrations of 0.075 and 0.15 µg/ml reactivated the propeptide either fully or partially. MIC of the D-P18 was 16µM at enzyme concentration of 0.15µg/ml and 32µM at enzyme concentration of 0.075µg/ml in S. aureus. The propeptide was reactivated and antibacterial activity observed against P. aeruginosa with MIC of 16µM at enzyme concentration of 0.15µg/ml. Addition of the profragment linker did not affect the antimicrobial activity of D-P18. This was observed after carrying comparison tests of neutrophil alone and D-P18. Peptides interfere with the membrane through electrostatic interactions, the profragment added to synthesized peptide, D-P18, did not hinder these interactions since addition of equimolar concentration of the profragment did not affect D-P18 antibacterial activity. Hemolytic assay to probe the toxicity of the proforms and mature D-P18 found that the mature D-P18 resulted in a dose-dependent lysis of erythrocytes at concentrations which ranged from 50-500µM whereas similar test with the proform of the peptide showed no erythrocytic effect at the same concentration range. This work further investigated the conformation changes in the proform and the mature peptide could be playing a role in the toxicity difference. 1H Nuclear magnetic resonance performed on the two peptides demonstrated that they did not form secondary structures in aqueous solution. Whereas D-P18 formed a left-handed short α-helical conformation from residues D-Ile8’ and D-Leu14’ in 50% 2, 2, 2-trifluoroethano (TFE-d3) - water mixed solvent system, the proform (Ac-E4-A3-D-P18) had an α-helical conformation between D-Lys8 and D-Ile15 residues. Hence the differences observed between the proforms and the mature form of the synthetic peptide, D-LP18 may be attributed from the NMR studies to the reduction of the net charge. Host defence peptides are ubiquitous in the living systems. Their role in the innate immune system cannot be underestimated. This work has reported a synthetic propeptide, D-P18 which is able to circumvent host’s protein degradation machinery. The propeptide has been shown to be processed by a neutrophil elastase, an abundant serine protease in the epithelial lining of cystic fibrosis lung. The prepeptide is able to be cleaved by the enzyme due to the presence of a trialanine linker which is its substrate. By using an enantiomer of L-P18, the peptide is able to reach the target pathogens in cystic fibrosis without the interference from protein degrading enzymes. This paper has reported a novel synthetic propeptide with in vitro activity against representative pathogens in a cystic fibrosis lung. The studies conclusions and results are based on an in vitro study, however is of importance to carry out assessment of the activities of the propeptide in in vivo systems. This will greatly consolidates in vitro studies on important factors such as the toxicity of the peptide and its proform (Ac-E4-A3-D-P18). Work cited [1]. Bowdish, D.M., Davidson, D.J. and Hancock, R.E. “A Re-evaluation of the Role of Host Defence Peptides in Mammalian Immunity.” Current Protein and Peptide Science. 6.1(2005): 35-51. [2]. Desgranges, S., Prieult, F., Daly, A., Lydon, J., Brennan, M., Rai, D.K.,Subasinghage, A.P., Hewage, C.M., Cryan, S., Greene, C., McElvaney, N.G., Smyth, T.P., Fitzgerald-Hughes, D., Humphreys, H. and Devocelle, M. “In Vitro Activities of Synthetic Host Defense Propeptides Processed by Neutrophil Elastase against Cystic Fibrosis Pathogens” Antimicrobial Agents and Chemotherapy, 55.5 (2011):2487-2489. [3]. Boat, T.F., Welsh, M.J. and Beaudet, A.L. “Cystic fibrosis. In: The Metabolic Basisof Inherited Disease. (Edited by; Scriver, C.R., Beaudet A.L., Sly, W.S. and Valle D.). New York: McGraw-Hill, (1989):2649-80. [4]. Kerem, B., Rommens, J.M., Buchanan, J.A., et al. “Identification of the cystic fibrosis gene: genetic analysis.” Science 245(1989): 1073 -1080. [5]. McElvaney, N.G., Nakamura, H., Birrer, P., et al. “Modulation of airway inflammation in cystic fi brosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor.” J Clin Invest 90(1992):1296 -1301. [6]. Smallman, L.A., Hill, S.L. and Stockley, R.A. ‘Reduction of ciliary beat frequency in vitro by sputum from patients with bronchiectasis: a serine proteinase effect.” Thorax, 39 (1984): 663-667. [7]. Breuer, R., Christensen, T.G., Niles, R.M., et al. “Human neutrophil elastase causes glycoconjugate release from the epithelial cell surface of hamster trachea in organ culture.” Am Rev Respir Dis 139(1989):779-782 [8]. Linde, A., B. Wachter,B., Hőner, O.P., L. Dib, L., Ross, C., Tamayo, A.R., F. Blecha, F. and Melgarejo, T. “Natural History of Innate Host Defense Peptides” Probiotics and Antimicro. Prot. 1(2009):97-112 [9]. 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