| Porcine lactoferricin-20 (LFP-20) is a 20-amino acid residue of cationic antimicrobial peptide derived from the porcine lactoferrin N terminus. In this study, we designed the analogs using LFP-20 as a template and screening the antibacterial activity, hemolytic activity and cytotoxicity to peripheral blood mononuclear cells of analogs. Furthermore, the comparative studies of modified peptides LF-2, LF-4, LF-6 and LFP-20 in the membrane mechanism of action were conducted. The protective effects of analogs against E.coli were investigated using mouse model with E.coli infection in thigh and abdominal cavity. Then, effects of analogs on the immune function of health and infected mouse with Escherichia.coli infection were discussed. Moreover, the analog LF-6 was expressioned successfully using E.coli expression system and Pichia pastoris expression system. The main results are as follows:1. Molecular improvement and screening of porcine lactoferricinEight analogs were obtained by strategies of removing the intermolecular disulfide bonds, changing the proportion of hydrophobic and aromatic amino acids of LFP-20 based on the analysis of LFP-20 physicochemical properties, amino acid composition and structure prediction. Compared to LFP-20, the analogs LF-2, LF-4 and LF-6 exhibited 2～64 times increased antimicrobial activities against gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella choleraesuis, Salmonella typhimurium, and 2-8 times increased antibacterial activities against gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis. Among three analogs, LF-6 showed the best antimicrobial activities. LF-2, LF-4 and LF-6 did not induce increased hemolytic activity significantly (p> 0.05) to human and porcine erythrocyte below 32μg/mL compared to LFP-20. Furthermore, the proliferations of human and porcine peripheral blood mononuclear cells (PBMCs) were not influenced significantly (p> 0.05) by LF-2, LF-4 and LF-6 below 50μg/mL. Except 8μg/mL and 16μg/mL LF-4, the cytotoxicities of LF-2, LF-4 and LF-6 to PBMCs were not also increased significantly (p> 0.05) below 32μg/mL compared to LFP-20.2. Membrane mechanism of action of porcine lactoferricin and analogsThe effects of LFP-20 and its analogs LF-2, LF-4, LF-6 on E.coli and S.aureus morphology observed by SEM and TEM showed four peptides led to different degrees of membrane protrusions or damage, the cell wall and membrane abruption, cell contents leak, decreasing cytoplasmic electron density at 1×MIC after 30 min incubation. These results indicated that cytoplasmic membrane of bacteria is one of the important targets of LFP-20 and its analogs LF-2. LF-4, LF-6.At the same concentration, three analogs LF-2, LF-4, LF-6 caused stronger membrane depolarization of E.coli and S.aureus than LFP-20, which more likely to lead to ion channel formation on cell membrane. Compared to LFP-20, three analogs enhanced the E.coli outer membrane permeability significantly (p<0.05) at 8～32μg/mL. Among four peptides, LF-6 led to the most rapid and obvious inner membrane permeability of E.coli. LFP-20, LF-4, LF-6 had similar inhibiting rate (Imax) to binding ability between DPX and LPS, which were 68%,56% and 51% respectively. However, the concentrations of LF-4 and LF-6 required for 50% inhibition rate (I50) to binding ability between DPX and LPS were significantly lower than LFP-20, which were 7.96μg/mL and 6.91μg/mL respectively, and the I50 of LFP-20 was 17.30μg/mL. Although the ability of replacement DPX molecule in DPX-LPS of LF-4, LF-6 weaker than LFP-20, peptides concentration required to achieve maximum binding with LPS for LF-4 and LF-6 lower significantly than LFP-20. Four antimicrobial peptides had similar damage potential to the liposome membrane at 16μg/mL and 32μg/mL. However, compared with LFP-20, three analogs presented a more damage potential on liposome membrane PC: PG (1:1) and PG at 64μg/mL, causing more than 40% calcein release. These results revealed that membrane disruption mechanism is the major mechanism of action of LFP-20 and three analogs against E.coli and S.aureus. Compared with LFP-20, the enhanced antimicrobial activity of LF-2, LF-4 and LF-6 were related to the increased cytoplasmic membrane depolarization, inner and outer membrane permeability, more easily combined with LPS and the damage potential to liposome membrane.3. Protective effects of porcine lactoferricin and analogs on E.coli infected mouseBased on the study of acute toxicity of LFP-20 and analogs LF-2, LF-6 to ICR mouse, the mouse thigh and abdominal infection model with E.coli K88 were established to compare the protective ability LFP-20 and LF- 2, LF-6 against E.coli infection. The results showed that LD50 of LFP-20 and LF-2, LF-6 were 34.25 mg/kg,16.54 mg/kg and 29.52 mg/kg. The effects of LFP-20 and LF-2, LF-6 to mouse thigh-infection model showed that three peptides have a protective effect on mouse infected with E.coli K.88 at 2 mg/kg and 8 mg/kg. In addition to 2 mg/kg LFP-20, another five doses of the peptides significantly reduced the number of in thigh muscle (p<0.05). The inhibitory effect of 8 mg/kg LF-6 in thigh muscle was the most significant (p<0.05), and the number of E.coli in homogenate of thigh muscle was 3.85±0.24 (lg CFU/g). The effects of LFP-20 and LF-2, LF-6 on mouse abdominal infection model showed that 2 mg/kg and 8 mg/kg of the three peptides could inhibit the infection of E.coli K88 significantly (p<0.05) of peritoneal fluid, liver and mesenteric lymph nodes in mouse model. The inhibitory effect of 8 mg/kg LF-6 was the most significant (p<0.05) compared with E.coli control, and the number of E.coli in homogenate of peritoneal fluid, liver and mesenteric lymph nodes were 1.18±0.10 (lg CFU/mL),3.85±0.24 (lg CFU/g) and 3.00±0.15 (lg CFU/g), respectively. The number of E.coli, Lactobacillus and Bifidobacterium in cecum contents of E.coli control were 5.57±0.16 (lg CFU/g),6.32±0.09 (lg CFU/g) and 5.54±0.17 (lg CFU/g). The number of E.coli in cecum contents of peptides groups (in addition to 2 mg/kg LFP-20) were significantly lower than E.coli control (p<0.05), and the number of Lactobacillus and Bifidobacterium (in addition to 8 mg/kg LFP-20) were significantly higher than E.coli control (p<0.05). The number of E.coli in fecal of six peptides groups were significantly lower than E.coli control (p<0.05), and the number of Lactobacillus of 2 mg/kg LF-2 were significantly higher than E.coli control (p<0.05). The number of Bifidobacterium of six peptides groups were significantly higher than E.coli control (p<0.05). The results revealed that the ability to resist E.coli K88 infection of mouse could be enhanced by LFP-20 and analogs LF-2, LF-6 through bacteriostasis in vivo and improve the number decrease of Lactobacillus and Bifidobacterium caused by intestinal infection at the same time. LF-2 and LF-6 held the better bacteriostasis than LFP-20 in vivo.4. Effects of porcine Iactoferricin and analogs on immune function in mouseThe results of effect of LFP-20 and analogs LF-2, LF-6 on immune function in ICR mice showed that 2 mg/kg LF-2 and LF-6 increased the mouse thymus index(p<.05); 8 mg/kg LF-2 and LF-6 reduced mice thymus index (p<0.05) and spleen index (p<0.05) compared with control mouse. The percentage of peripheral blood lymphocytes and the total number of peripheral white blood cells were also increased significantly (p<0.05) by 8 mg/kg LF-6 and 2 mg/kg LF-6 respectively. The proportion of peripheral blood CD3+CD4+ lymphocytes of mouse were reduced significantly (p<0.05) by LFP-20, LF-2, LF-6 at 2 mg/kg and LF-2, LF-6 at 8 mg/kg.2 mg/kg LF-2, LF-6, and 8 mg/kg LF-2 could increased the proportion of peripheral blood B cells significantly (p<0.05), but had no significant influence on the number of NK cells. The LPS stimulation index and ConA stimulation index to spleen lymphocytes in 8 mg/kg LF-2 and 2 mg/kg LF-6 group were significant (p<0.05) higher than that of control group.The results of effect of LFP-20 and analogs LF-2, LF-6 on immune function in ICR mice infected with E.coli K88 showed that the thymus index of mouse in E.coli control was decreased significantly compared to the health control. The thymus index of 2 mg/kg LF-2, LF-6, and 8 mg/kg LFP-20, LF-2 groups had increasing trend compared to E.coli control. Compared with health control, the percentage of peripheral blood lymphocytes of E.coli control had increasing trend (p> 0.05). But the LFP-20 and analogs LF-2, LF-6 decreased the percentage of peripheral blood lymphocytes significantly (p<0.05) compared to E.coli control. The peripheral blood CD3+CD8+numbers of E.coli control was significantly decreased and the ratio of CD3+CD4+and CD3+CD8+increased significantly (p<0.05). The number of NK cells was also reduced significantly (p<0.05) by E.coli infection. The number of peripheral blood CD3+CD8+cells of 2 mg/kg of LFP-20, LF-6, and 8 mg/kg of the LFP-20, LF-2 groups were significantly higher than that of E.coli control (p<0.05), and 8 mg/kg LF-2 increased the proportion of peripheral blood NK cells significantly (p<0.05). The LPS and ConA stimulation index to mouse spleen lymphocytes of E.coli control were significantly higher than that of health control (p<0.05). Six peptides decreased the LPS and ConA stimulation index significantly (p<0.05) compared to E.coli control. The LPS stimulation index to mouse spleen lymphocytes of 2 mg/kg and 8 mg/kg LFP-20 the ConA stimulation index of 2 mg/kg LFP-20 was significantly higher (p<0.05) than E.coli control. Compared with health control, the antibody formation ability of spleen cells in mouse of E.coli control did not influenced significantly, but 2 mg/kg of three antimicrobial peptides and 8 mg/kg LF-2, LF-6 enhanced the antibody formation ability of spleen cells significantly (p<0.05). The gene expression levels of cytokines IL-1, IL-10, TNF-a and chemokine MCP-1, MIP-la of E.coli control were significantly higher than those of health control (p<0.05). LF-2 reduced the gene expression level of MCP-1 and increased the gene expression level of MIP-1α, IL-10 significantly (p<0.05) compared with E.coli control.2 mg/kg LF-6 reduced the MCP-1, MIP-la and IL-10 gene expression levels significantly (p<0.05), and 8 mg/kg LF-6 reduced the MCP-1, MIP-la, IL-1, IL-10 and TNF-a gene expression level significantly (p<0.05) compared with E.coli control.5. The recombinant expression of porcine Iactoferricin analogThe recombinant expression of analog LF-6 in E.coli. The primers were designed according to E.coli codon preference and amino acid sequence of LF-6, then target genes EK-LF-6 and TEV-LF-6 were amplified successfully by overlap PCR. After constructed the target genes into expression vector pET32a, the recombinant E. coli strain BL21 (DE3) pLysS- pET32a- TEV- LF-6 and BL21 (DE3) pLysS- pET32a- EK- LF-6 were obtained. The expression of fusion protein Trx-EK-LF-6 and Trx-TEV-LF-6 were detected through IPTG induction and SDS-PAGE analysis. Expression levels of soluble fusion protein Trx-TEV-LF-6 and Trx-EK-LF-6 were 40.60 mg/L and 42.13 mg/L respectively by proteins determination using Bradford method, gel analysis and calculated. The theory expression levels of LF-6 were 5.59 mg/L and 6.13 mg/L after two fusion proteins cleaved by protease. The two fusion proteins cleaved by TEV and EK after purification. Results showed that the cleavage efficiency of TEV higher than EK. The antimicrobial activies of cleavage products were detected using agar hole diffusion method after freeze-drying of the products. Results showed that cleavage products of TEV had antimicrobial activities against both E.coli ATCC25922 and E.coli K88, but exhibited higher potential to against E.coli K88. These indicated that the Gly residue in the LF-6 N terminal less affected the antimicrobial activity of LF-6 after TEV cleavageThe recombinant expression of analog LF-6 in P. pastoris. The primers were designed according to yeast codon preference and amino acid sequence of LF-6, then target gene were amplified successfully by overlap PCR. After constructed the target gene into expression vector pPICZaA, recombinant plasmid PICZa-LF-6 were transformed into protease-deficient yeast strain SMD1168. Recombinant strain P.pastoris SMD1168-pPICZaA-LF-6 was obtained. The expression of antimicrobial peptides LF-6 was induced by methanol and the fermentation supernatant were detected. Tricine-SDS-PAGE results showed that the expression level of target peptide LF-6 reached a high level after the recombinant strain induced for 6 days. LF-6 expression level was measured using the concentrated fermentation supernatant by Bradford method, which was 20 mg/L. The expression product exhibited the antimicrobial activities against E.coli K88In summary, we obstained the analogs LF-2 and LF-6 with increased antimicrobial activity against gram-negative and gram-positive bacteria through the molecular improvement of LFP-20. The hemolysis rate and cytotoxicity to PBMCs of LF-2 and LF-6 were not enhanced significantly at a certain range of concentration. Compared to template peptide LFP-20, analogs LF-2 and LF-6 showed stronger damage potential on bacterial cytoplasmic membrane and enhanced capacity to resist the infection of mice with E.coli K88 in vivo. LF-2 and LF-6 could also improve the number reduction of Bifidobacterium and Lactobacillus caused by infection. LF-2 and LF-6 were able to regulate the immune functions through changing the thymus index, proportion of B cells in peripheral blood, transformation efficiency of spleen lymphocyte, and control the abnormal changes in immune parameters ofanimal model caused by E.coli infection. In addition, analog LF-6 were expressed successfully using E. coli and P. pastoris expression system. The theory expression levels of LF-6 were 5.59 mg/L and 20mg/L repectively, and products exhibited obvious antibacterial activity against E.coli.
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