动物源抗菌肽的分子改良及其对猪肠道上皮屏障功能的保护作用研究
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摘要
抗菌肽因其广谱抗菌活性和不易产生耐药性等特点引起了人们的高度重视。本研究通过对人源抗菌肽LL-37,猪源抗菌肽Protegrin-1(PG-1), PMAP-23, Porcine Lactoferricin (LfcinP),牛源抗菌肽Bovine Lactoferricin (LfcinB), indolicidin (IN),蛇源抗菌肽Cathelicidin-BF (C-BF),蛙源抗菌肽Plastrin-OG1(P-OG1),虫源抗菌肽cceropin A (CA), cecropinP1(CP1)等十种不同动物源抗菌肽的抗菌活性、杀菌机制、对真核细胞的细胞毒性及与抗生素的协同作用进行比较研究,在此基础上通过杂合改良抗菌肽以获得具有高抗菌活性、低细胞毒性且具有不易诱导细菌产生耐药性的杀菌机制的抗菌肽；进一步利用猪小肠上皮细胞为模型研究抗菌肽对猪肠道上皮屏障功能的保护作用。围绕上述研究主要开展以下三方面试验：
试验一不同动物源抗菌肽的抗菌活性、杀菌机制和细胞毒性的比较研究
对不同动物源抗菌肽抗菌活性的比较中,PG-1和C-BF具有广谱抗菌活性,对革兰氏阴性菌特别是致腹泻型大肠杆菌具有较好的作用,最小抑菌浓度(MIC)在16-64μg/mL,与金霉素的抗菌活性相当；时间动态曲线结果表明C-BF在MIC浓度下5min即可有效杀灭致病菌,而金霉素则需要1-2h。另外,CP1对革兰氏阴性菌、IN对革兰氏阳性菌也具有较好的抗菌活性。同时比较了抗菌肽和饲用抗生素对益生菌的抗菌活性,所试抗菌肽对嗜酸乳杆菌和双歧杆菌不具有抑制作用,而金霉素、新霉素具有较大的抑制作用。抗菌肽与饲用抗生素的联合应用结果表明，金霉素与PG-1或C-BF对致腹泻型大肠杆菌的FIC值均小于0.5,表现为协同作用,且能有效降低金霉素的作用浓度,MIC值是金霉素单用时的1／8到1／2。
进一步对不同动物源抗菌肽的杀菌机制进行研究,利用扫描电镜和透射电镜观察了不同动物源抗菌肽对细菌细胞膜的作用,并通过DNA结合试验、蛋白质合成抑制试验及ATP泄露试验研究了抗菌肽细胞内作用靶点。结果表明大多数抗菌肽作用后使大肠杆菌和金黄色葡萄球菌表面出现了不同程度的凸起、凹陷或破损,部分可见内容物外泄。另外,PG-1和C-BF具有与DNA结合、促进ATP泄露等胞内作用机制。抗菌肽与抗生素对肠道益生菌的作用机制比较结果表明,大多数抗菌肽对益生菌细胞膜没有影响,而金霉素、新霉素对猪嗜酸乳杆菌的细胞膜具有破坏作用,使细菌破碎,结构不完整。上述结果提示,细菌细胞膜可能是抗菌肽作用的主要靶点,同时一些抗菌肽存在细胞内作用机制。
通过溶血率试验、细胞增殖率试验及LDH释放率试验对不同动物源抗菌肽对真核细胞毒性试验结果表明,LfcinB、LfcinP、C-BF、PMAP-23、CP1和CA对猪红细胞和外周血单个核细胞(PBMCs)具有很低的细胞毒性；而LL-37、PG-1、IN和P-OG1呈现剂量依赖效应,即在高浓度(128-256μg/mL)时具有较大的细胞毒性。结果提示,部分抗菌肽具有较高的细胞毒性,在作为新型抗菌制剂研制前应考虑其安全性。
试验二杂合抗菌肽的分子设计及结构和生物学活性研究
在上述研究基础上,以安全性好但抗菌活性低的CA和LfcinB,及抗菌活性好但细胞毒性高的PG-1为模板肽,在分析其理化性质、氨基酸组成和结构预测的基础上,采用“分子拼接”的设计方法,选择LfcinB活性中心序列LfcinB (4-9)和CA的N端α螺旋结构片段CA(1-8),与PG-1的β折叠主体结构进行杂合,得到两个杂合肽LfcinB (4-9)-PG-1(5-17)(简写为LB-PG)和CA (1-8)-PG-1(1-16)(简写为CA-PG),以期在保持PG-1原有抗菌活性的基础上降低其细胞毒性。通过结构预测及圆二色谱法分析杂合肽LB-PG和CA-PG在模拟细胞膜环境即在SDS和TFE溶剂中的结构特征,结果表明杂合肽LB-PG和CA-PG均为与模板肽PG-1相似的β折叠结构。
抗菌活性和细胞毒性试验结果表明,杂合肽LB-PG对革兰氏阴性菌的抗菌活性较好,与模板肽PG-1相当,对猪红细胞和PBMCs的细胞毒性比PG-1显著降低；CA-PG扩大了对革兰氏阳性菌的抗菌谱,也显著降低了对猪红细胞和PBMCs的细胞毒性。杀菌机制试验结果表明细菌细胞膜是杂合肽作用的主要靶点,菌体表面出现不同程度的凸起或凹陷,且使大肠杆菌菌体变长；LB-PG同时具有与细菌DNA结合的胞内作用机制。
试验三抗菌肽对仔猪肠道上皮屏障功能的保护作用及机制研究
以试验一筛选得到的抗菌肽C-BF和试验二改良得到的杂合肽LB-PG为研究对象,以猪空肠上皮细胞系IPEC-J2为模型,利用透射电镜、Ussing chamber、免疫荧光等方法,在大肠杆菌内毒素LPS刺激下研究了抗菌肽对猪肠道上皮屏障功能的保护作用及其可能机制。透射电镜观察结果表明,自然分化21-24天的IPEC-J2细胞和细胞之间之处可见紧密连接复合物,如紧密连接,桥粒连接,细胞间空隙等。C-BF和LB-PG处理24h后对紧密连接结构没有明显影响,紧密连接结构清晰,深度约0.2-0.5μm。Ussing chamber检测表明加入C-BF和LB-PG处理后,在上样30min、60min、90min和120min后IPEC-J2的跨上皮电阻(TER)分别提高了58.2%、91.9%、86.5%、220%和55.8%、122%、79.3%、249%,即促进了肠道上皮屏障功能。
LPS刺激后破坏了IPEC-J2的紧密连接结构,紧密连接打开；预先用C-BF和LB-PG处理24h再加入LPS,紧密连接结构保持清晰完整,说明抗菌肽能够缓解LPS对IPEC-J2紧密连接的破坏作用。Ussing chamber试验结果表明,LPS诱导在短时间(15min)内迅速降低了TER73.7%,破坏了肠上皮屏障的完整性；预先用C-BF和LB-PG处理24h再加入LPS, IPEC-J2的TER与对照组相比差异不显著。说明抗菌肽能够缓解LPS导致的IPEC-J2的屏障功能的破坏。上述结果提示抗菌肽对LPS诱导破坏的肠道上皮屏障功能具有一定的保护作用。
在上述研究基础上,进一步探讨抗菌肽对组成紧密连接重要的成分即紧密连接蛋白表达的影响,利用实时荧光定量PCR和Western blot检测抗菌肽对紧密连接蛋白Claudin、 Occludin、ZO-1和ZO-2的表达的影响,利用激光共聚焦显微镜观察抗菌肽对紧密连接蛋白在IPEC-J2分布的影响,并通过加入MAP激酶两条级联信号通路MEK和p38的抑制剂研究抗菌肽作用的信号转导通路。qRT-PCR及Western blot结果表明,LPS刺激显著降低了IPEC-J2细胞紧密连接蛋白的表达。预先用C-BF处理24h再加入LPS,紧密连接蛋白表达水平与对照组相比差异不显著,提示抗菌肽能够缓解LPS诱导的IPEC-J2紧密连接蛋白表达下降。同时,免疫荧光结果表明,LPS刺激使紧密连接蛋白Occludin和ZO-1在IPEC-J2细胞边缘和细胞质分布异常且含量减少,预先用C-BF处理24h再加入LPS,Occludin和ZO-1在IPEC-J2细胞边缘和细胞质的分布和表达恢复到对照组水平,提示抗菌肽能够缓解LPS诱导的IPEC-J2紧密连接蛋白分布异常。MAP激酶级联信号中的两条通路MEK1/2和p38的抑制剂即U0126和SB203580均显著降低了C-BF作用后IPEC-J2中紧密连接蛋白的表达。结果提示C-BF可能通过MAP激酶信号转导通路促进IPEC-J2中紧密连接蛋白的表达,进而缓解由LPS刺激造成的应激反应,保护肠道上皮屏障功能。
上述研究一方面为研制新型抗菌免疫制剂的研制提供了优良的候选肽,同时在了解抗菌肽对肠道屏障功能保护作用的基础上,为通过营养手段提高抗菌肽表达、改善猪的健康水平提供了研究思路,这对研发新型抗菌免疫制剂,有效替代或减少抗生素作为饲料添加剂的使用、提高畜产品安全性具有重要意义。
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 AMPs
PG-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 activities
Since 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 mechanism
In 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.
试验一不同动物源抗菌肽的抗菌活性、杀菌机制和细胞毒性的比较研究
对不同动物源抗菌肽抗菌活性的比较中,PG-1和C-BF具有广谱抗菌活性,对革兰氏阴性菌特别是致腹泻型大肠杆菌具有较好的作用,最小抑菌浓度(MIC)在16-64μg/mL,与金霉素的抗菌活性相当；时间动态曲线结果表明C-BF在MIC浓度下5min即可有效杀灭致病菌,而金霉素则需要1-2h。另外,CP1对革兰氏阴性菌、IN对革兰氏阳性菌也具有较好的抗菌活性。同时比较了抗菌肽和饲用抗生素对益生菌的抗菌活性,所试抗菌肽对嗜酸乳杆菌和双歧杆菌不具有抑制作用,而金霉素、新霉素具有较大的抑制作用。抗菌肽与饲用抗生素的联合应用结果表明，金霉素与PG-1或C-BF对致腹泻型大肠杆菌的FIC值均小于0.5,表现为协同作用,且能有效降低金霉素的作用浓度,MIC值是金霉素单用时的1／8到1／2。
进一步对不同动物源抗菌肽的杀菌机制进行研究,利用扫描电镜和透射电镜观察了不同动物源抗菌肽对细菌细胞膜的作用,并通过DNA结合试验、蛋白质合成抑制试验及ATP泄露试验研究了抗菌肽细胞内作用靶点。结果表明大多数抗菌肽作用后使大肠杆菌和金黄色葡萄球菌表面出现了不同程度的凸起、凹陷或破损,部分可见内容物外泄。另外,PG-1和C-BF具有与DNA结合、促进ATP泄露等胞内作用机制。抗菌肽与抗生素对肠道益生菌的作用机制比较结果表明,大多数抗菌肽对益生菌细胞膜没有影响,而金霉素、新霉素对猪嗜酸乳杆菌的细胞膜具有破坏作用,使细菌破碎,结构不完整。上述结果提示,细菌细胞膜可能是抗菌肽作用的主要靶点,同时一些抗菌肽存在细胞内作用机制。
通过溶血率试验、细胞增殖率试验及LDH释放率试验对不同动物源抗菌肽对真核细胞毒性试验结果表明,LfcinB、LfcinP、C-BF、PMAP-23、CP1和CA对猪红细胞和外周血单个核细胞(PBMCs)具有很低的细胞毒性；而LL-37、PG-1、IN和P-OG1呈现剂量依赖效应,即在高浓度(128-256μg/mL)时具有较大的细胞毒性。结果提示,部分抗菌肽具有较高的细胞毒性,在作为新型抗菌制剂研制前应考虑其安全性。
试验二杂合抗菌肽的分子设计及结构和生物学活性研究
在上述研究基础上,以安全性好但抗菌活性低的CA和LfcinB,及抗菌活性好但细胞毒性高的PG-1为模板肽,在分析其理化性质、氨基酸组成和结构预测的基础上,采用“分子拼接”的设计方法,选择LfcinB活性中心序列LfcinB (4-9)和CA的N端α螺旋结构片段CA(1-8),与PG-1的β折叠主体结构进行杂合,得到两个杂合肽LfcinB (4-9)-PG-1(5-17)(简写为LB-PG)和CA (1-8)-PG-1(1-16)(简写为CA-PG),以期在保持PG-1原有抗菌活性的基础上降低其细胞毒性。通过结构预测及圆二色谱法分析杂合肽LB-PG和CA-PG在模拟细胞膜环境即在SDS和TFE溶剂中的结构特征,结果表明杂合肽LB-PG和CA-PG均为与模板肽PG-1相似的β折叠结构。
抗菌活性和细胞毒性试验结果表明,杂合肽LB-PG对革兰氏阴性菌的抗菌活性较好,与模板肽PG-1相当,对猪红细胞和PBMCs的细胞毒性比PG-1显著降低；CA-PG扩大了对革兰氏阳性菌的抗菌谱,也显著降低了对猪红细胞和PBMCs的细胞毒性。杀菌机制试验结果表明细菌细胞膜是杂合肽作用的主要靶点,菌体表面出现不同程度的凸起或凹陷,且使大肠杆菌菌体变长；LB-PG同时具有与细菌DNA结合的胞内作用机制。
试验三抗菌肽对仔猪肠道上皮屏障功能的保护作用及机制研究
以试验一筛选得到的抗菌肽C-BF和试验二改良得到的杂合肽LB-PG为研究对象,以猪空肠上皮细胞系IPEC-J2为模型,利用透射电镜、Ussing chamber、免疫荧光等方法,在大肠杆菌内毒素LPS刺激下研究了抗菌肽对猪肠道上皮屏障功能的保护作用及其可能机制。透射电镜观察结果表明,自然分化21-24天的IPEC-J2细胞和细胞之间之处可见紧密连接复合物,如紧密连接,桥粒连接,细胞间空隙等。C-BF和LB-PG处理24h后对紧密连接结构没有明显影响,紧密连接结构清晰,深度约0.2-0.5μm。Ussing chamber检测表明加入C-BF和LB-PG处理后,在上样30min、60min、90min和120min后IPEC-J2的跨上皮电阻(TER)分别提高了58.2%、91.9%、86.5%、220%和55.8%、122%、79.3%、249%,即促进了肠道上皮屏障功能。
LPS刺激后破坏了IPEC-J2的紧密连接结构,紧密连接打开；预先用C-BF和LB-PG处理24h再加入LPS,紧密连接结构保持清晰完整,说明抗菌肽能够缓解LPS对IPEC-J2紧密连接的破坏作用。Ussing chamber试验结果表明,LPS诱导在短时间(15min)内迅速降低了TER73.7%,破坏了肠上皮屏障的完整性；预先用C-BF和LB-PG处理24h再加入LPS, IPEC-J2的TER与对照组相比差异不显著。说明抗菌肽能够缓解LPS导致的IPEC-J2的屏障功能的破坏。上述结果提示抗菌肽对LPS诱导破坏的肠道上皮屏障功能具有一定的保护作用。
在上述研究基础上,进一步探讨抗菌肽对组成紧密连接重要的成分即紧密连接蛋白表达的影响,利用实时荧光定量PCR和Western blot检测抗菌肽对紧密连接蛋白Claudin、 Occludin、ZO-1和ZO-2的表达的影响,利用激光共聚焦显微镜观察抗菌肽对紧密连接蛋白在IPEC-J2分布的影响,并通过加入MAP激酶两条级联信号通路MEK和p38的抑制剂研究抗菌肽作用的信号转导通路。qRT-PCR及Western blot结果表明,LPS刺激显著降低了IPEC-J2细胞紧密连接蛋白的表达。预先用C-BF处理24h再加入LPS,紧密连接蛋白表达水平与对照组相比差异不显著,提示抗菌肽能够缓解LPS诱导的IPEC-J2紧密连接蛋白表达下降。同时,免疫荧光结果表明,LPS刺激使紧密连接蛋白Occludin和ZO-1在IPEC-J2细胞边缘和细胞质分布异常且含量减少,预先用C-BF处理24h再加入LPS,Occludin和ZO-1在IPEC-J2细胞边缘和细胞质的分布和表达恢复到对照组水平,提示抗菌肽能够缓解LPS诱导的IPEC-J2紧密连接蛋白分布异常。MAP激酶级联信号中的两条通路MEK1/2和p38的抑制剂即U0126和SB203580均显著降低了C-BF作用后IPEC-J2中紧密连接蛋白的表达。结果提示C-BF可能通过MAP激酶信号转导通路促进IPEC-J2中紧密连接蛋白的表达,进而缓解由LPS刺激造成的应激反应,保护肠道上皮屏障功能。
上述研究一方面为研制新型抗菌免疫制剂的研制提供了优良的候选肽,同时在了解抗菌肽对肠道屏障功能保护作用的基础上,为通过营养手段提高抗菌肽表达、改善猪的健康水平提供了研究思路,这对研发新型抗菌免疫制剂,有效替代或减少抗生素作为饲料添加剂的使用、提高畜产品安全性具有重要意义。
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 AMPs
PG-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 activities
Since 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 mechanism
In 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.
引文
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