Design And Screening Of Potent Antibacterial Peptides Based On Human Defensin 5 And A Structure-activity Study

Due to the attenuated immune defense, human beings exposed to irradiation are susceptible to the infection of exogenous pathogens. Along with the deepening crisis of drug resistance, these people are seriously threatened by resistant bacteria. Because intestine is sensitive to radiation, and because there are abundant bacteria locating at the enteric cavity, irradiation often induces enterogenic infections. Enteric defensin is a group of amphiphilic peptides in the line of mammalian innate immunity, which exerts potent antibacterial activity. It is difficult for microbes to evolve to resist enteric defensin. Human defensin 5(HD5) is a Paneth cell-secreted antibacterial peptide, which can protect host from the invasion of pathogens by selectively suppressing pathogenic bacteria and regulating the intestinal microbiota. It is a promising candidate to treat infections caused by irradiation. Previously, we investigated the alteration and function of HD5 in radiation induced enterogenic infections. Moreover, we prepared highly purified HD5 by gene recombination and expression. However, due to the shieding effects of salt ions and anionic proteins, the bacterial killing of HD5 is limited in vivo. Accordingly, we try to modify the amino acid sequence of HD5 by structure-activity studies, aiming at enhancing the antibacterial activity and improving the bioavailability of HD5.HD5 is composed by 32 amino acids(ATCYCRTGRCARESLSGVCEISGRLYRLCC R). Cys residues are connected as Cys1-6/Cys2-4/Cys3-5, which confers a ?-sheet conformation to HD5. HD5 has multivalent binding ability, and it can polymerize in a dose-dependent manner, which magnifies the antibacterial activity of monomeric peptide. Recently, it was demonstrated that human ? defensin 1(HBD1) was reduced by thioredoxin(Trx) in intestine, and the reduced linear peptide was more efficient than HBD1 in bacterial killing. Using a serious of molecular and biological experiments, we first confirm that reduced HD5 does exist in vivo. Nevertheless, different from HBD1, reduction impairs the antibacterial activity of HD5. There is an antibacterial active region formed by Arg25, Leu26, Tyr27, and Arg28, in the C-terminus of HD5, where the positively charged Arg residues are responsible for the electrostatic interaction with bacteria, and the hydrophobic Leu and the aromatic Tyr contribute to the membrane destruction. Other than the conserved Glu in salt bridge, Glu21 is the sole negatively charged residue in HD5, and it locates nearby the active region. We conduct a charge-reversal mutation by replacing Glu21 with Arg and discover that E21 R mutation attenuates the self-association but enhances the antibacterial activity of HD5. Furthermore, we substitute Thr7 of E21R-HD5 to Arg and find that T7 R mutation not only improves the bacterial killing but benefits the self-association of monomeric E21R-HD5, and T7E21R-HD5 exerts potent antibacterial efficiency in saline solution and in serum.The specific disulfide pairings can protect HD5 from the degradation of trypsin, which is instrumental to the stability of the peptide in vivo. Because linear peptides are easier to be synthesized and because the content of trypsin in local tissue, such as cornea, skin, and respiratory tract, is relatively low, topical administration of linear antibacterial peptides to treat infection caused by resistant bacteria turns to be a hotspot. Combined with the effects of disulfide reduction and Arg mutation on the antibacterial activity of HD5, we design a mutant with only one disulfide bond and potent efficiency against multi-drug resistant(MDR) Acinetobacter baumannii(A. baumannii). Besides the peptidic design and screening, we also conduct biochemical analysis to complete the structure-activity study. Additionally, we investigate the underlying antibacterial mechanism of these peptides. The main results and conclusions of this study are as below:1. Using acid urea-polyacrylamide gel electrophoresis(AU-PAGE) Western blot and matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF), we first detect oxidized and reduced HD5 in the luminal fluid of small intestine. In vitro experiments support that intestinal thioredoxin(Trx) can catalyze the reduction of oxidized HD5.2. Reduction impairs the antibacterial activity of oxidized HD5. Results of the bacterial surface potential detection, Biolayer interferometry(BLI), NPN uptake, E.coli ML35 inner membrane permeabilization assay, and flow cytometry show that reduced HD5 is weaker than oxidized HD5 in bacterial attraction, lipid A binding, outer and inner membrane penetration, and inducing the depolarization of bacterial membrane, thus demonstrating the underlying machenism of the bactericidal attenuation.3. Reduction improves the LPS-neutralization of oxidized HD5. Results of the circular dichroism(CD), isothermal titration calorimetry(ITC), LBP-blocking assay, immunofluorescence, and quantitative PCR show that reduced HD5 has the superiority in structural flexibility, which benefits to binding LPS and blocking the interaction between LPS and LBP, thus lowering the initiation of the TLR4-NF-?B pathway and suppressing the release of inflammatory factors.4. Using virtual colony count assay, we discover that substituting Glu21 to Arg(E21R) can enhance the antibacterial activity of HD5. Results of BLI, NPN uptake, and E.coli ML35 inner membrane permeabilization assay show that E21R-HD5 is stronger than HD5 in lipid A and lipoteichoic acid(LTA) binding and outer/inner membrane destruction, thus demonstrating the underlying machenism of the bactericidal enhancement.5. ITC result shows that E21 R mutation impairs the self-association of HD5. Using X-ray single crystal diffraction, we determine the crystal structure of E21R-HD5 and submit it to Protein Data Bank(PDB). The number of E21R-HD5 in PDB is 4RBX. We find that E21R-HD5 is a monomer. Disturbing the self-association is detrimental to the bacterial killing of defensin. Though impairing the self-association, E21 R mutation markedly enhances the antibacterial activity of HD5, which indicates that Arg mutation can compensate for the functional attenuation caused by conformational deficiency.6. Crystal visualization analysis shows that Thr7, Glu21, Ser23, and Gly24 are four residues nearby the C-terminal active region of HD5. Using virtual colony count assay, we find that when the residue is substituted to Arg(T7R, E21 R, S23 R, G24R), the antibacterial activities of the mutants are: E21R-HD5 > T7R-HD5 > S23R-HD5 ≈ G24R-HD5。7. Using virtual colony count assay and ITC, we discover that T7 R mutation not only enhances the antibacterial activity but improves the self-association of E21R-HD5. We employ X-ray single crystal diffraction to determine the crystal structure of T7E21R-HD5 and submit it to PDB(4RBX). T7E21R-HD5 is an atypical dimmer. This is the first data demonstrating that Arg mutation can benefit the self-association of monomeic peptide.8. Results of BLI, NPN uptake, and E.coli ML35 inner membrane permeabilization assay show that T7E21R-HD5 is stronger than E21R-HD5 in lipid A and LTA binding and outer/inner membrane destruction, thus demonstrating the underlying machenism of the bactericidal enhancement.9. T7E21R-HD5 can overcome the shieding effects of salt ions, and it is efficient in the presence of serum. Moreover, T7E21R-HD5 is harmless to erythrocyte, and it can resist the degradation of trypsin.10. We employ Ala mutation and find that removing disulfide bonds and retaining only one disulfide bond both impair the antibacterial activity of HD5 against MDR A. baumannii. The mutant(HM3) with Cys2-4 disulfide bond is stronger than other peptides in bacterial killing. Substituting the non-electropositive and non-hydrophobic residues of HM3 to Arg creates a potent antibacterial peptide(HM5).11. Using colony count assay, we find that HM5 kills MDR A. baumannii in dose and time dependent manners, and it is efficient in the condition of high concentrations of saline solution. Results of BLI, NPN uptake, and flow cytometry show that HM5 is stronger than HD5 in lipid A binding, bacterial outer membrane penetration, and inducing the membrane depolarization, thus demonstrating the machenism of the bactericidal enhancement.12. We conduct gene recombinant expression and prepare highly purified A. baumannii outer membrane protein A(OMPA). OMPA is detrimental to host cells. Our data show that HM5 is stronger than HD5 in OMPA-neutralization. BLI provides that HM5 is more potent than HD5 in OMPA-binding, which we think is related to the improved OMPA-neutralization ablility of HM5.