Structure-functional analyses of bacterial cell agglutinating antimicrobial peptides

• Main Author: Sinha, Sheetal
• Other Authors: Surajit Bhattacharyya
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences::Biophysics
• Online Access: https://hdl.handle.net/10356/84291; http://hdl.handle.net/10220/46666
• Institution: Nanyang Technological University

Innate antimicrobial peptides have served as the first line of defense against pathogens throughout human history. Host defense cationic AMPs are broad spectrum antimicrobials that can kill bacteria, viruses and fungi. On the basis of their modes of action, AMPs can be broadly classified into three categories – membrane permeabilizing AMPs, AMPs having intracellular targets and cell agglutinating AMPs. Bacterial cell death by membrane permeabilization has been well perceived. In the recent years, increasing number of studies are conducted on AMPs targeting intracellular components. However, very few AMPs are known to agglutinate bacterial cells. Moreover, the mechanism is of agglutination is poorly understood. Multiple binding sites in AMPs for outer membrane lipopolysaccharides or cell wall peptidoglycans are pivotal for agglutination. However, a complete understanding of the mechanism is required to develop novel antimicrobials with agglutination as a mode of action. In this thesis, the first atomic resolution structure of thanatin, a cell agglutinating peptide in complex with LPS has been reported. Thanatin assumes a four stranded antiparallel beta-sheet conformation in a ‘head-tail’ dimeric topology. In contrast, thanatin in free solution assumes an antiparallel beta-hairpin conformation. Dimerization of thanatin provides multiple LPS binding sites needed to facilitate agglutination. Furthermore, dimeric thanatin displays higher cationicity and hydrophobicity. Various gain-in function and loss-in function mutation were synthesized to determine the structure-activity correlation. Biophysical analyses of thanatin and mutant peptides were conducted to determine LPS/peptide binding constant. Perturbation of LPS micelles in the presence of these peptides was also analysed using biophysical and NMR experiments. Finally, MD simulations were conducted on thanatin dimer/LPS complex to understand the mode of LPS recognition and agglutination. Structural changes in thanatin in presence of zwitterionic DPC micelles were also analysed. Unlike in complex with LPS, thanatin adopts a monomeric topology in presence of DPC micelles. Paramagnetic relaxation enhancement experiments were carried out to examine the interaction of thanatin with DPC micelles. The mechanistic insights on bacterial cell agglutination can be utilized to develop antimicrobials with alternative mode of action. viii Furin is a serine endoprotease which is ubiquitously found in mammals. Its prodomain is known to possess anti-cancerous properties. In this report, the antimicrobial property of a furin prodomain derived peptide has been reported and elucidated for the first time. Various truncations from furin prodomain were screened for their antimicrobial activity. YR26, derived from the central region of the prodomain displays a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. To determine the structure-activity correlation, various truncations were made from the C-terminus of the peptide. YR26 completely loses its activity on truncating more than six residues from C-terminus. Furthermore, the active peptides were found to be non-hemolytic and non-toxic to mammalian cells in vitro. To gain insights on the mode of action, membrane permeabilization assays and other biophysical experiments were conducted. The active AMPs were found to completely permeabilize outer membrane but were not potent in permeabilizing the inner membrane of E. coli cells. Moreover, the experiments indicated a higher order of association of E. coli cells/LPS micelles in the presence of these peptides. Interaction studies of POPC-POPG and POPC liposomes with the active prodomain derived peptides strongly indicate agglutination as a mode of action. In order to gain further insights on the mechanism of agglutination, 3D structure of YR26 in presence of SDS micelles was determined using solution state NMR spectroscopy. YR26 adopts an anti-parallel alpha-helical conformation with two distinct helices attached with an extended loop. The two anti-parallel alpha-helical region provides multiple LPS binding site to facilitate cell agglutination. YR26 can be further used a template for the discovery of novel antimicrobial drug.