Molecular Design Of Animal Antimicrobial Peptides And Study Their Protective Effects On Intestine Epithelial Barrier Function

Antimicrobial peptides (AMPs) have attracted considerable attention for their broad-spectrum antimicrobial activity and less possibility to cause bacterial resistance. In this study, antibacterial activity, mechanisms of action and cytotoxicity of ten different animal AMPs including Protegrin-1(PG-1), PMAP-23, Porcine Lactoferricin (LfcinP) from swine, Bovine Lactoferricin (LfcinB), indolicidin (IN) from bovine, Cathelicidin-BF (C-BF) from snake, Plastrin-OG1(P-OG1) from frog, cceropin A (CA), cecropin P1(CP1) from insect were investigated, and hybrid peptides with higher antimicrobial activity and lower cytotoxicity were obtained based on above results. Furthermore, the protective effect of AMPs on swine intestinal epithelial barrier function using an epithelial cell line originating from the jejunum of pig (IPEC-J2) was studied. The main results were as follows:1Comparative study of the antibacterial activity, mechanism of action and cytotoxicity of animal AMPsPG-1and C-BF were the most active peptides not only against Gram-positive bacteria, but also Gram-negative bacteria, especially Enteropathogenic Escherichia coli strains isolated from fecal samples of weaning piglets with diarrhea. In addition, the bactericidal activity of these two peptides was almost equal to aureomycin. Killing kinetics showed that PG-1and C-BF were more rapid and efficient bactericide than aureomycin. Besides, CP1and IN demonstrated high inhibitor activity towards Gram-negative and Gram-positive bacteria, respectively. We also tested the inhibitor action of these peptides and antibiotics to intestine beneficial bacteria. Most tested peptides showed no adverse effects on Bifidobacterium suis and Lactobacillus acidophilus, while aureomycin and neomycin showed a marked inhibitory effect. Combination effect of AMPs and antibiotics showed synergistic effects between PG-1and aureomycin and C-BF and aureomycin against EPEC E. coli078:K80and the MICs of the antibiotics obviously decrease while the peptides were added.The permeabilize effect of AMPs on bacteria cell membrane was studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the potential intracellular targets of AMPs were investigated by DNA binding, protein synthesis inhibition and ATP release assays. Most of the tested AMPs were found to permeabilize bacterial membranes, and cytoplasmic contents were leaking out of the cells in some cases. Furthermore, intracellular targets such as binding to DNA and increasing ATP release maybe exist for PG-1and C-BF. Meanwhile, we compared the mechanism of AMPs and antibiotics on beneficial bacteria. Most AMPs were not disrupting membrane of Bifidobacterium suis and Lactobacillus acidophilus, while aureomycin and neomycin kill Bifidobacterium suis by lysing the cell membrane. From the data above, it is concluded that membrane permeability and damage should be considered as the principal step, intracellular targets were also important for AMPs.Cytotoxicity tests showed that LfcinB, LfcinP, C-BF, PMAP-23, CP1and CA exhibited the lower cytotoxic effects, while LL-37, PG-1, indolicidin and OG1displayed higher cytotoxic activity among128-256μg/mL. Such cytotoxicity of some AMPs should be decreased before they are used as new antibacterial agents.2Design of novel hybrid β-sheet peptides with enhanced cell specificity and analysis their structures and biological activitiesSince PG-1had considerable antimicrobial activity but high cytotoxicitiy, while LfcinB and CA had lower cytotoxicity as described above, we designed novel hybrid peptides LB-PG and CA-PG, according to the combination the P-fold fragment of protegrin-1(1-16) or (5-17) with active center of bovine lactoferricin (4-9) or N-terminal of cecropin A (1-8) in order to reduce cytotoxicity of PG-1as bactericidal capacity can be better saved. The structure analysis of hybrid peptides was performed by bioinformatics programs and circular dichroism (CD) in the membrane-mimicking environment, such as SDS and TFE. Results showed LB-PG and CA-PG still adopted the predominant β-sheet conformation as PG-1.The antimicrobial and cytotoxicity assays demonstrated that LB-PG displayed similar antimicrobial activities compared to the natural peptide PG-1and relatively low hemolytic activity, showing a2-4fold decrease at each concentration when compared to PG-1. CA-PG showed higher inhibitory effect than CA and extended antibacterial spectra, especially against Gram-positive bacteria, and also decreased cytotoxicity significantly. Mechanism study elucidated that these peptides killed microbial cells by penetrating the cell membrane. Gel retardation study revealed the LB-PG had higher affinity to DNA, indicating one possible intracellular bactericidal mechanism.3Study of the protective effect of AMPs on intestinal epithelial barrier function and potential mechanismIn this work, we evaluated the ability of C-BF and LB-PG in upregulating tight junction (TJ) protein expression and improving the epithelial permeability of porcine intestinal epithelial cell line (IPEC-J2) under normal condition and LPS stimulation, using TEM, Ussing Chamber and immunofluorescence. The selective MEK1/2and p38inhibitors were applied in determining the signaling pathway.TEM revealed that from day21onwards, well formed junctional complexes in IPEC-J2such as tight junctions, desmosomes, zonula adherens and intercellular spaces. After co-culture with C-BF and LB-PG for24h, these junctional complexes seem similar to those seen in untreated IPEC-J2cells. Meanwhile, compared with IPEC-J2monolayers alone, incubation with C-BF or LB-PG for24h significantly increased the TER. Exposed to LPS markedly opened the TJ structure of IPEC-J2. In addition, LPS treatment also decreased of TER in a short time. While, C-BF and LB-PG treatment markedly attenuated the tight junction structural abnormalities induced by LPS and significantly relieved the decreased of TER in IPEC-J2monolayer. The results above confirm that AMPs could improve or protect intestinal epithelial barrier function.Incubation with C-BF for24h significantly increased the expression of Occludin and ZO-1of IPEC-J2at mRNA and protein levels. Although basolateral LPS stimulation markedly inhibited the expression of TJ proteins, co-culture with C-BF significantly restored the expression of Occludin and ZO-1at the mRNA and protein levels. TJ proteins Occludin and ZO-1were typically expressed around the cell border and developed relatively tidy network when cells reached confluence using Immunofluorescence. Compared with the IPEC-J2cells alone adding C-BF significantly increased the expression of Occludin and ZO-1around IPEC-J2border. LPS markedly disrupted the TJ network of IPEC-J2and the normal distribution of Occludin and ZO-1was replaced by an aberrant pericellular location. C-BF treatment markedly attenuated the tight junctional abnormality induced by LPS. In order to investigate the potential signaling pathway, selective MEK1/2and p38inhibitors were used to detect the activated and basal forms of MAP kinase. Both inhibitors pretreatment could completely abolish the stimulatory effect of C-BF on TJ protein expression. Results indicated C-BF protects the intestinal epithelial barrier function throuth upregulating TJ mRNA and protein expression and maybe regulated by MAP kinase signaling pathway.In conclusion, our research implicated that piglet immunity could be improved by upregulation of secretory expression of porcine AMPs, and some AMPs have potential for development as antimicrobial agents for substitution of feed antibiotics in pig diets.