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Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

Development and Characterization of Polymeric Peptides for Antibody Tagging of Bacterial Targets

Author(s): Fresthel Monica M. Climacosa*, Ruby Anne N. King, Bobbie Marie M. Santos and Salvador Eugenio C. Caoili

Volume 27, Issue 10, 2020

Page: [962 - 970] Pages: 9

DOI: 10.2174/0929866527666200427212940

Price: $65


Background: Microbe-Binding Peptides (MBPs) are currently being investigated to address the problem of antimicrobial resistance. Strategies enhancing their antimicrobial activity have been developed, including peptide dimerization. Here, we present an alternative approach based on peptide polymerization, yielding hapten-labelled polymeric MBPs that mediate tagging of bacteria with anti-hapten antibodies, for enhanced immune recognition by host phagocytes.

Methods: C-terminally amidated analogs of the bacterial-binding peptide IIGGR were synthesized, with or without addition of cysteine residues at both N- and C-termini. Peptides were subjected to oxidizing conditions in a dimethyl-sulfoxide/water solvent system, and polymerization was demonstrated using SDS-PAGE. Peptides were then N-terminally labelled with a trinitrophenyl (TNP) group using trinitrobenzene sulfonate (TNBS). Binding to representative bacteria was demonstrated by ELISA using anti-TNP antibodies and was quantified as half-maximal effective concentration (EC50). Minimum Inhibitory Concentration (MIC) and concentration yielding 50% hemolysis (H50) were estimated. Neutrophil phagocytic index was determined for TNP-labelled polymeric bacterial- binding peptide (Pbac) with anti-TNP antibodies and/or serum complement.

Results: Polydisperse Pbac was synthesized. EC50 was lower for Pbac than for the corresponding monomeric form (Mbac), for both Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. MIC and H50 were >250μg/mL for both Pbac and Mbac. A complement-independent increase in neutrophil phagocytic index was observed for E. coli treated with TNP-labelled Pbac in conjunction with anti-TNP antibodies.

Conclusion: Our data suggest that hapten-labelled polymeric bacterial-binding peptides may easily be produced from even crude synthetic oligopeptide precursors, and that such bacterial-binding peptides in conjunction with cognate anti-hapten antibodies can enhance immune recognition of bacteria by host phagocytes.

Keywords: Antimicrobial peptides, disulfides, polymerization, haptens, chemically programmable immunity, antibody-dependent cellular phagocytosis, opsonization.

Graphical Abstract
Walker, J.M. Antimicrobial Peptides: Methods and Protocols 2010.
Ruiz, J.; Calderon, J.; Rondón-Villarreal, P.; Torres, R. Analysis of structure and hemolytic activity relationships of Antimicrobial Peptides (AMPs). In: Advances in Intelligent Systems and Computing; Castillo, L.; Cristancho, M.; Isaza, G.; Pinzón, A.; Rodríguez, J., Eds.; Springer: Cham, 2014; Vol. 232, pp. 253-258.
Bahar, A.A.; Ren, D. Antimicrobial peptides. Pharmaceuticals, 2019, 6(12), 1543-1575.
Lorenzon, E.N.; Piccoli, J.P.; Santos-Filho, N.A.; Cilli, E.M. Dimerization of antimicrobial peptides: A promising strategy to enhance antimicrobial peptide activity. Protein Pept. Lett., 2019, 26(2), 98-107.
Caoili, S.E.C. Antibodies, synthetic peptides and related constructs for planetary health based on green chemistry in the Anthropocene. Future Sci. OA, 2018, 4(3), FSO275.
[] [PMID: 29568564]
Rader, C. Chemically programmed antibodies. Trends Biotechnol., 2014, 32(4), 186-197.
[] [PMID: 24630478]
Mullis, K. Chemically Programmable Immunity. U.S. Patent 8,591,910, November 26.
Popkov, M.; Gonzalez, B.; Sinha, S.C.; Barbas, C.F., III Instant immunity through chemically programmable vaccination and covalent self-assembly. Proc. Natl. Acad. Sci. USA, 2009, 106(11), 4378-4383.
[] [PMID: 19255430]
Hillyer, C.; Josephson, C.; Blajchman, M.; Vostal, J.; Epstein, J.; Goodman, J. Bacterial contamination of blood components: Risks, strategies, and regulation: Joint ASH and AABB Educational Session in Transfusion Medicine. Hematology (Am. Soc. Hematol. Educ. Program), 2003, 2003(1), 575-589.
Wiegand, I.; Hilpert, K.; Hancock, R.E.W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc., 2008, 3(2), 163-175.
[] [PMID: 18274517]
Dagur, P.K.; McCoy, J.P. Collection, storage, and preparation of human blood cells. In: Current Protocols in Cytometry; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2015; 73, pp. 5.1.1-5.1.16.
Bangalore, N.; Travis, J.; Onunka, V.; Pohl, J.; Shafer, W. Identification of the primary antimicrobial domains in human neutrophil cathepsin G. J. Biol. Chem., 1990, 265, 13584-13588.
Wang, G.; Li, X.; Wang, Z. APD3: The antimicrobial peptide database as a tool for research and education. Nucleic Acids Res., 2016, 44(D1), D1087-D1093.
[] [PMID: 26602694]
Waghu, F.H.; Barai, R.S.; Gurung, P.; Idicula-Thomas, S. CAMPR3: A database on sequences, structures and signatures of antimicrobial peptides. Nucleic Acids Res., 2016, 44(D1), D1094-D1097.
[] [PMID: 26467475]
Fan, L.; Sun, J.; Zhou, M.; Zhou, J.; Lao, X.; Zheng, H.; Xu, H. DRAMP: A comprehensive data repository of antimicrobial peptides. Sci. Rep., 2016, 6, 24482.
[] [PMID: 27075512]
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680-685.
[] [PMID: 5432063]
M07-A10: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Tenth Edition 2015, 35(2), 1-87.
Drevets, D.A.; Canono, B.P.; Campbell, P.A. Measurement of bacterial ingestion and killing by macrophages. Curr. Protoc. Immunol, 2015, 109(1), 14.6.1-14.6.7.
Miyasaki, K.T.; Bodeau, A.L.; Pohl, J.; Shafer, W.M. Bactericidal activities of synthetic human leukocyte cathepsin G-derived antibiotic peptides and congeners against Actinobacillus actinomycetemcomitans and Capnocytophaga sputigena. Antimicrob. Agents Chemother., 1993, 37(12), 2710-2715.
[] [PMID: 8109940]
Rüegg, S.J.; Jungi, T.W. Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of high-affinity and low-affinity receptors for IgG by using different erythroid target cells. Immunology, 1988, 63(3), 513-520.
[PMID: 2965100]
McAleer, M.; Sim, R. The Complement System.Activators and Inhibitors of Complement; Sim, R.B., Ed.; Springer Science Business Media: Oxford, UK, 1993, p. 242.
Pauly, D.; Nagel, B.M.; Reinders, J.; Killian, T.; Wulf, M.; Ackermann, S.; Ehrenstein, B.; Zipfel, P.F.; Skerka, C.; Weber, B.H.F. A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples. PLoS One, 2014, 9(5), e96371.
[] [PMID: 24797388]
Oddo, A.; Hansen, P.R. Hemolytic activity of antimicrobial peptides. In: Antimicrobial Peptides: Methods and Protocols, Methods in Molecular Biology; Humana Press: New York, NY, 2017; pp. 427-435.
Palmer, M. Cholesterol and the activity of bacterial toxins. FEMS Microbiol. Lett., 2004, 238(2), 281-289.
[] [PMID: 15358412]
Zipperer, A.; Kretschmer, D. Cytotoxicity Assays as Predictors of the Safety and Efficacy of Antimicrobial Agents. In: Antibiotics: Methods and Protocols, Methods in Molecular Biology; Springer Science+Business Media New: New York,, 2017; 1520, pp. 107-118.
Kofler, H.; Schnegg, I.; Geley, S.; Helmberg, A.; Varga, J.M.; Kofler, R. Mechanism of allergic cross-reactions--III. cDNA cloning and variable-region sequence analysis of two IgE antibodies specific for trinitrophenyl. Mol. Immunol., 1992, 29(2), 161-166.
[] [PMID: 1542295]
Di, L. Strategic approaches to optimizing peptide ADME properties. AAPS J., 2015, 17(1), 134-143.
[] [PMID: 25366889]
Mülhaupt, R. Green polymer chemistry and bio-based plastics: Dreams and reality. Macromol. Chem. Phys., 2013, 214(2), 159-174.

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