Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576 bp an

No evidence of expression of two classes of natural antibiotics (cathelicidins and defensins) in a sample of platypus milk

Marsupial neonates are born without a fully functioning immune system, and are known to be protected in part by natural antimicrobial peptides present in their mother's milk. Monotreme neonates hatch at a similar stage in development, and it has been hypothesised that their survival in a non-sterile burrow also relies on the presence of natural antibiotics in their mother's milk. Here we review the field of monotreme lactation and the antimicrobial peptide complement of the platypus (Ornithorhynchus anatinus). Using reverse transcriptasepolymerase chain reaction of milk cell RNA from a sample of platypus milk, we found no evidence for the expression of cathelicidins or defensins in the milk. This was unexpected. We hypothesise that these natural antibiotics may instead be produced by the young platypuses themselves.

Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation.

Influences of porcine circo-, arteriviruses and cathelicidins in plasmacytoid dendritic cell-derived interferon-alpha responses

Type I interferons (IFNs), mainly IFN-α/β play a crucial role in innate defense against viruses. In addition to their direct antiviral activity, type I IFNs have antitumoral and immunomodulatory effects. Although all cells are virtually able to induce IFN-α, the plasmacytoid dendritic cell (pDC) subset represents the ultimate producers of IFN-α as well as other proinflammatory cytokines. Due to the specific expression of TLR7 and TLR9 recognizing single-stranded (ss) RNA and unmethylated CpG motifs respectively, pDCs can secrete up to 1000 times more IFN-α than any cellular types. Additionally, it is well known that several cytokines including type I and II IFNs, Flt3-L, IL-4 and GM-CSF favor pDC-derived IFN-α responses to unmethylated CpG motifs. In a first step, we aimed to characterize and clarify the interactions of two porcine viruses with pDCs. The double-stranded DNA replicative forms of porcine circovirus type 2 (PCV2) were demonstrated to inhibit CpG-induced IFN- α by pDCs. Our study showed that none of the cytokines known to enhance pDC responsiveness can counter-regulate the PCV2-mediated inhibition of IFN-α induced by CpG, albeit IFN-γ significantly reduced the level of inhibition. Interestingly, the presence of IFN-γ enabled pDCs to induce IFN-α to low doses of PCV2. We also noted that after DNase treatment, PCV2 preparations were still able to stimulate pDCs. These data suggest that encapsulated viral ssDNA promotes the induction of IFN-α in pDCs treated with IFN-γ whereas free DNA, presumably as double-stranded forms, was responsible for inhibiting pDC responses. Regarding PRRSV, it has been reported that North American isolates did not induce and even inhibited IFN-α response in pDCs. However, PRRSV infection was also shown to lead to an induction of IFN-α in the serum and in the lungs suggesting that certain cells are responsive to the virus. Contrasting to previous reports we found that numerous PRRSV isolates directly induced IFN-α in pDCs. This response was still observed after UV-inactivation of viruses and required TLR7 signaling. The inhibition of CpG-induced IFN-α was weak and strain dependent, again contrasting with a previous report. We also observed that IFN-γ and IL-4 enhanced IFN-α response to two prototype strains, VR-2332 and LVP23. In summary, we demonstrated that both PCV2 and PRRSV promote IFN-α secretion in pDCs in vitro suggesting that IFN-α detected in PCV2- or PRRSV-infected animal might originate from pDCs. On the other hand, PRRSV replication is restricted to the macrophage (MΦ) lineage. These innate immune cells represent a heterogeneous population which can be induce to “classical” (M1) and “alternative” (M2) activated MΦ acquiring inflammatory or “wound-healing” functional properties, respectively. Nonetheless, little is known about the effect of polarization into M1 or M2 and the susceptibility of these cells to PRRSV. Thus, we examined the impact of cytokine on MΦ polarization into M1 or M2. Infections of these cells by several PRRSV isolates enabled the discrimination of PRRSV isolate in a genotype- and irulencedependent manner in M1 and IFN-β-activated MΦ. In contrast, the expression of PRRSV nucleocapsid in M2 or inactivated MΦ was indistinguishable among the PRRSV isolates tested. In the last part of my Thesis, we investigated the influence of three synthetic porcine cathelicidin peptides for their ability to deliver nucleic acid to pDCs. We reported that all cathelicidins tested can complex and quickly deliver nucleic acids resulting in IFN-α induction. Moreover, we show that the typical α- helical amphipathic conformation is required to mediate killing of bacteria but not for inducing IFN-α secretion by pDCs. Furthermore, we found that E.coli treated with one of these cathelicidins is able to induce significantly higher levels of IFN-α compared to a non-sense version of the peptide. These data suggest that cathelicidins could influence the immune response in a two-step process. First, these peptides target bacteria leading to cell lysis. In turn, cathelicidins form complexes and deliver extracellular microbial nucleic acids released into pDCs. These pDC-derived IFN-α responses could be of particular relevance in driving the adaptive immune responses against microbial infections.

Porcine cathelicidins efficiently complex and deliver nucleic acids to plasmacytoid dendritic cells and can thereby mediate bacteria-induced IFN-α responses.

Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine-rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide-DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid-complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections.

Seminal plasma proteome of eletroejaculated Bos indicus bulls

The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4±2.3 and 64±3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 D-isomarase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization.

眼镜蛇科 cathelicidin 基因的分子克隆及活性研究

Cathelicidins 是天然免疫系统中的一种带正电的宿主防御肽段，它们广泛地 分布在哺乳类及其他一些物种如鱼类，鸟类中。它们均包含保守的前肽区和多变 的C-末端成熟抗菌肽区域，该抗菌肽区域无论是在种间还是种内都不保守。 我们首次分别从爬行纲眼镜蛇科的眼镜蛇，金环蛇，眼镜王蛇三种毒蛇的毒 腺cDNA 文库中克隆了3 个cathelicidin 编码序列。所克隆到的序列编码的开放 阅读框架均长576bp，编码191 个氨基酸残基组成的蛋白前体。从cDNA 开放阅 读框推导得到的毒蛇cathelicidin 都含有22 个氨基酸残基组成的信号肽， 135 个 氨基酸残基组成的cathelin 保守区域以及34 个氨基酸残基组成的成熟肽区域。 与哺乳类中的cathelicidin 基因的高度多样性不同，来源于3 种毒蛇的cathelicidin 基因在核酸和蛋白水平都比较保守。结构分析表明，以上3 种毒蛇的cathelicidin 成熟肽由第157 位的Val 被elastase 切割而产生。采用化学合成法合成推导得到 的眼镜王蛇的cathelicidin（OH-CATH）。在1% NaCl 的浓度下，该合成肽对测试 的多种细菌具有很强的抑菌活性，MIC 值为1-20 μg/ml。与此同时，即使当浓度 高达200 μg/ml 时，该合成的肽段对人的红细胞依然没有溶血活性。对脊椎动物 的cathelicidin 遗传进化树分析发现毒蛇类的cathelicidin 聚在一起。从进化上看， 蛇的cathelicidin 与来源于小鼠、大鼠、兔的中性粒细胞颗粒蛋白更接近。毒蛇 的cathelicidin 可能为新药开发提供了一个很好的模板。

Novel cathelicidin-derived antimicrobial peptides from Equus asinus

In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 mu g center dot mL-1. EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 mu g center dot mL-1. CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl2, EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

金环蛇抗菌肽cathelicidin-BF的纯化、基因克隆、结构与功能分析及抗菌机理研究

Cathelicidins是动物体内一个具有多功能的抗菌肽家族，目前仅在哺乳类、鸟类、爬行类和鱼类中有发现。Cathelicidins具有广谱的抗微生物活性，不但对普通革兰氏阳性细菌、革兰氏阴性细菌、真菌以及病毒具有非常强的活性，而且对许多临床耐药微生物同样具有作用。除此之外，cathelicidins具有许多其他生物学活性，如对多种免疫细胞具有趋化作用、诱导肥大细胞脱粒和组织胺释放、调节巨噬细胞转录、促进伤口愈合、诱导血管发生、诱导变异细胞系细胞凋亡和淋巴细胞活化等。 金环蛇（Bungarus fasciatus）属于眼镜蛇科（Elapinae）环蛇属（Bungarus），是一种具有前沟牙的毒蛇，广泛分布于我国广东、广西、福建、江西、海南及云南南部。 在本论文中，我们对金环蛇体内cathelicidins家族抗菌肽cathelicidin-BF进行了一系列研究。 通过凝胶过滤、阳离子交换和反相高压液相三步从金环蛇蛇毒冻干粉中分离纯化得到金环蛇cathelicidins家族抗菌肽，命名为cathelicidin-BF。Edman降解法测定其氨基酸序列为KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF，由30个氨基酸残基组成，ESI-MS测得其分子量为3637.5 Da。 构建了金环蛇毒腺cDNA文库，从中克隆得到了编码cathelicidin-BF前体的cDNA序列。该cDNA序列长度为750 bp，由此推断出的cathelicidin-BF前体由191个氨基酸残基组成，包括信号肽区、保守的cathelin区和成熟肽区三部分。用RT-PCR的方法对cathelicidin-BF的组织表达情况进行了研究，结果表明cathelicidin-BF在金环蛇胃、气管、皮肤、肌肉、心脏、肾脏、肺、脑、小肠、脾脏、肝脏、卵巢、毒腺中均有表达，但各组织表达量存在差异。进化分析表明，金环蛇cathelicidin-BF与鸭嘴兽CATH-3在系统进化树中独立成簇，表明金环蛇与鸭嘴兽有着一定的亲缘关系，这为鸭嘴兽在动物进化中分类地位的确定提供了参考资料。 对cathelicidin-BF可能具有的各种生物学活性进行了研究。Cathelicidin-BF具有广谱的抗微生物活性，对革兰氏阳性细菌、革兰氏阴性细菌和真菌均有活性，Cathelicidins是动物体内一个具有多功能的抗菌肽家族，目前仅在哺乳类、鸟类、爬行类和鱼类中有发现。Cathelicidins具有广谱的抗微生物活性，不但对普通革兰氏阳性细菌、革兰氏阴性细菌、真菌以及病毒具有非常强的活性，而且对许多临床耐药微生物同样具有作用。除此之外，cathelicidins具有许多其他生物学活性，如对多种免疫细胞具有趋化作用、诱导肥大细胞脱粒和组织胺释放、调节巨噬细胞转录、促进伤口愈合、诱导血管发生、诱导变异细胞系细胞凋亡和淋巴细胞活化等。 金环蛇（Bungarus fasciatus）属于眼镜蛇科（Elapinae）环蛇属（Bungarus），是一种具有前沟牙的毒蛇，广泛分布于我国广东、广西、福建、江西、海南及云南南部。 在本论文中，我们对金环蛇体内cathelicidins家族抗菌肽cathelicidin-BF进行了一系列研究。 通过凝胶过滤、阳离子交换和反相高压液相三步从金环蛇蛇毒冻干粉中分离纯化得到金环蛇cathelicidins家族抗菌肽，命名为cathelicidin-BF。Edman降解法测定其氨基酸序列为KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF，由30个氨基酸残基组成，ESI-MS测得其分子量为3637.5 Da。 构建了金环蛇毒腺cDNA文库，从中克隆得到了编码cathelicidin-BF前体的cDNA序列。该cDNA序列长度为750 bp，由此推断出的cathelicidin-BF前体由191个氨基酸残基组成，包括信号肽区、保守的cathelin区和成熟肽区三部分。用RT-PCR的方法对cathelicidin-BF的组织表达情况进行了研究，结果表明cathelicidin-BF在金环蛇胃、气管、皮肤、肌肉、心脏、肾脏、肺、脑、小肠、脾脏、肝脏、卵巢、毒腺中均有表达，但各组织表达量存在差异。进化分析表明，金环蛇cathelicidin-BF与鸭嘴兽CATH-3在系统进化树中独立成簇，表明金环蛇与鸭嘴兽有着一定的亲缘关系，这为鸭嘴兽在动物进化中分类地位的确定提供了参考资料。 对cathelicidin-BF可能具有的各种生物学活性进行了研究。Cathelicidin-BF具有广谱的抗微生物活性，对革兰氏阳性细菌、革兰氏阴性细菌和真菌均有活性，Cathelicidins是动物体内一个具有多功能的抗菌肽家族，目前仅在哺乳类、鸟类、爬行类和鱼类中有发现。Cathelicidins具有广谱的抗微生物活性，不但对普通革兰氏阳性细菌、革兰氏阴性细菌、真菌以及病毒具有非常强的活性，而且对许多临床耐药微生物同样具有作用。除此之外，cathelicidins具有许多其他生物学活性，如对多种免疫细胞具有趋化作用、诱导肥大细胞脱粒和组织胺释放、调节巨噬细胞转录、促进伤口愈合、诱导血管发生、诱导变异细胞系细胞凋亡和淋巴细胞活化等。 金环蛇（Bungarus fasciatus）属于眼镜蛇科（Elapinae）环蛇属（Bungarus），是一种具有前沟牙的毒蛇，广泛分布于我国广东、广西、福建、江西、海南及云南南部。 在本论文中，我们对金环蛇体内cathelicidins家族抗菌肽cathelicidin-BF进行了一系列研究。 通过凝胶过滤、阳离子交换和反相高压液相三步从金环蛇蛇毒冻干粉中分离纯化得到金环蛇cathelicidins家族抗菌肽，命名为cathelicidin-BF。Edman降解法测定其氨基酸序列为KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF，由30个氨基酸残基组成，ESI-MS测得其分子量为3637.5 Da。 构建了金环蛇毒腺cDNA文库，从中克隆得到了编码cathelicidin-BF前体的cDNA序列。该cDNA序列长度为750 bp，由此推断出的cathelicidin-BF前体由191个氨基酸残基组成，包括信号肽区、保守的cathelin区和成熟肽区三部分。用RT-PCR的方法对cathelicidin-BF的组织表达情况进行了研究，结果表明cathelicidin-BF在金环蛇胃、气管、皮肤、肌肉、心脏、肾脏、肺、脑、小肠、脾脏、肝脏、卵巢、毒腺中均有表达，但各组织表达量存在差异。进化分析表明，金环蛇cathelicidin-BF与鸭嘴兽CATH-3在系统进化树中独立成簇，表明金环蛇与鸭嘴兽有着一定的亲缘关系，这为鸭嘴兽在动物进化中分类地位的确定提供了参考资料。 对cathelicidin-BF可能具有的各种生物学活性进行了研究。Cathelicidin-BF具有广谱的抗微生物活性，对革兰氏阳性细菌、革兰氏阴性细菌和真菌均有活性，Cathelicidins是动物体内一个具有多功能的抗菌肽家族，目前仅在哺乳类、鸟类、爬行类和鱼类中有发现。Cathelicidins具有广谱的抗微生物活性，不但对普通革兰氏阳性细菌、革兰氏阴性细菌、真菌以及病毒具有非常强的活性，而且对许多临床耐药微生物同样具有作用。除此之外，cathelicidins具有许多其他生物学活性，如对多种免疫细胞具有趋化作用、诱导肥大细胞脱粒和组织胺释放、调节巨噬细胞转录、促进伤口愈合、诱导血管发生、诱导变异细胞系细胞凋亡和淋巴细胞活化等。 金环蛇（Bungarus fasciatus）属于眼镜蛇科（Elapinae）环蛇属（Bungarus），是一种具有前沟牙的毒蛇，广泛分布于我国广东、广西、福建、江西、海南及云南南部。 在本论文中，我们对金环蛇体内cathelicidins家族抗菌肽cathelicidin-BF进行了一系列研究。 通过凝胶过滤、阳离子交换和反相高压液相三步从金环蛇蛇毒冻干粉中分离纯化得到金环蛇cathelicidins家族抗菌肽，命名为cathelicidin-BF。Edman降解法测定其氨基酸序列为KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF，由30个氨基酸残基组成，ESI-MS测得其分子量为3637.5 Da。 构建了金环蛇毒腺cDNA文库，从中克隆得到了编码cathelicidin-BF前体的cDNA序列。该cDNA序列长度为750 bp，由此推断出的cathelicidin-BF前体由191个氨基酸残基组成，包括信号肽区、保守的cathelin区和成熟肽区三部分。用RT-PCR的方法对cathelicidin-BF的组织表达情况进行了研究，结果表明cathelicidin-BF在金环蛇胃、气管、皮肤、肌肉、心脏、肾脏、肺、脑、小肠、脾脏、肝脏、卵巢、毒腺中均有表达，但各组织表达量存在差异。进化分析表明，金环蛇cathelicidin-BF与鸭嘴兽CATH-3在系统进化树中独立成簇，表明金环蛇与鸭嘴兽有着一定的亲缘关系，这为鸭嘴兽在动物进化中分类地位的确定提供了参考资料。 对cathelicidin-BF可能具有的各种生物学活性进行了研究。Cathelicidin-BF具有广谱的抗微生物活性，对革兰氏阳性细菌、革兰氏阴性细菌和真菌均有活性，Cathelicidins是动物体内一个具有多功能的抗菌肽家族，目前仅在哺乳类、鸟类、爬行类和鱼类中有发现。Cathelicidins具有广谱的抗微生物活性，不但对普通革兰氏阳性细菌、革兰氏阴性细菌、真菌以及病毒具有非常强的活性，而且对许多临床耐药微生物同样具有作用。除此之外，cathelicidins具有许多其他生物学活性，如对多种免疫细胞具有趋化作用、诱导肥大细胞脱粒和组织胺释放、调节巨噬细胞转录、促进伤口愈合、诱导血管发生、诱导变异细胞系细胞凋亡和淋巴细胞活化等。 金环蛇（Bungarus fasciatus）属于眼镜蛇科（Elapinae）环蛇属（Bungarus），是一种具有前沟牙的毒蛇，广泛分布于我国广东、广西、福建、江西、海南及云南南部。 在本论文中，我们对金环蛇体内cathelicidins家族抗菌肽cathelicidin-BF进行了一系列研究。 通过凝胶过滤、阳离子交换和反相高压液相三步从金环蛇蛇毒冻干粉中分离纯化得到金环蛇cathelicidins家族抗菌肽，命名为cathelicidin-BF。Edman降解法测定其氨基酸序列为KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF，由30个氨基酸残基组成，ESI-MS测得其分子量为3637.5 Da。 构建了金环蛇毒腺cDNA文库，从中克隆得到了编码cathelicidin-BF前体的cDNA序列。该cDNA序列长度为750 bp，由此推断出的cathelicidin-BF前体由191个氨基酸残基组成，包括信号肽区、保守的cathelin区和成熟肽区三部分。用RT-PCR的方法对cathelicidin-BF的组织表达情况进行了研究，结果表明cathelicidin-BF在金环蛇胃、气管、皮肤、肌肉、心脏、肾脏、肺、脑、小肠、脾脏、肝脏、卵巢、毒腺中均有表达，但各组织表达量存在差异。进化分析表明，金环蛇cathelicidin-BF与鸭嘴兽CATH-3在系统进化树中独立成簇，表明金环蛇与鸭嘴兽有着一定的亲缘关系，这为鸭嘴兽在动物进化中分类地位的确定提供了参考资料。 对cathelicidin-BF可能具有的各种生物学活性进行了研究。Cathelicidin-BF具有广谱的抗微生物活性，对革兰氏阳性细菌、革兰氏阴性细菌和真菌均有活性，Cathelicidins是动物体内一个具有多功能的抗菌肽家族，目前仅在哺乳类、鸟类、爬行类和鱼类中有发现。Cathelicidins具有广谱的抗微生物活性，不但对普通革兰氏阳性细菌、革兰氏阴性细菌、真菌以及病毒具有非常强的活性，而且对许多临床耐药微生物同样具有作用。除此之外，cathelicidins具有许多其他生物学活性，如对多种免疫细胞具有趋化作用、诱导肥大细胞脱粒和组织胺释放、调节巨噬细胞转录、促进伤口愈合、诱导血管发生、诱导变异细胞系细胞凋亡和淋巴细胞活化等。 金环蛇（Bungarus fasciatus）属于眼镜蛇科（Elapinae）环蛇属（Bungarus），是一种具有前沟牙的毒蛇，广泛分布于我国广东、广西、福建、江西、海南及云南南部。 在本论文中，我们对金环蛇体内cathelicidins家族抗菌肽cathelicidin-BF进行了一系列研究。 通过凝胶过滤、阳离子交换和反相高压液相三步从金环蛇蛇毒冻干粉中分离纯化得到金环蛇cathelicidins家族抗菌肽，命名为cathelicidin-BF。Edman降解法测定其氨基酸序列为KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF，由30个氨基酸残基组成，ESI-MS测得其分子量为3637.5 Da。 构建了金环蛇毒腺cDNA文库，从中克隆得到了编码cathelicidin-BF前体的cDNA序列。该cDNA序列长度为750 bp，由此推断出的cathelicidin-BF前体由191个氨基酸残基组成，包括信号肽区、保守的cathelin区和成熟肽区三部分。用RT-PCR的方法对cathelicidin-BF的组织表达情况进行了研究，结果表明cathelicidin-BF在金环蛇胃、气管、皮肤、肌肉、心脏、肾脏、肺、脑、小肠、脾脏、肝脏、卵巢、毒腺中均有表达，但各组织表达量存在差异。进化分析表明，金环蛇cathelicidin-BF与鸭嘴兽CATH-3在系统进化树中独立成簇，表明金环蛇与鸭嘴兽有着一定的亲缘关系，这为鸭嘴兽在动物进化中分类地位的确定提供了参考资料。 对cathelicidin-BF可能具有的各种生物学活性进行了研究。Cathelicidin-BF具有广谱的抗微生物活性，对革兰氏阳性细菌、革兰氏阴性细菌和真菌均有活性，其中包括大量临床分离耐药菌株。Cathelicidin-BF对革兰氏阴性细菌的活性要强于革兰氏阳性细菌，此外对白色念珠菌、毕赤酵母和一些腐生性真菌也具有活性。Cathelicidin-BF的抗氧化活性、溶血活性、凝集素活性、丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂活性、细胞毒性、抗肿瘤活性均不明显。Cathelicidin-BF具有很强的肥大细胞脱颗粒活性。以上结果表明cathelicidin-BF在金环蛇抵御外界病原微生物侵袭的先天免疫反应中可能发挥了重要作用。 利用多种实验方法对cathelicidin-BF的结构和抗菌机理进行了研究。CD和NMR的实验结果表明，在亲水环境中，cathelicidin-BF为无规卷曲的构象；在疏水或模拟细菌细胞质膜的环境中，cathelicidin-BF的N-末端区域具有典型的两亲性α-螺旋构象。杀菌动力学实验结果表明，cathelicidin-BF杀菌作用极其迅速，在浓度大于1×MIC时，在1 min内即可杀死所有细菌，且其杀菌作用是致死性的。扫描电镜结果表明，经过cathelicidin-BF处理的细菌细胞形状发生明显改变，细胞膨胀变形，表面出现大量囊泡状结构。大量细菌细胞破裂溶解，内容物外泄。综合以上结果我们推测：cathelicidin-BF在亲水的环境中为无规卷曲的结构，当通过静电相互作用吸附到细菌细胞质膜上后，由于环境疏水性的增加其N-端转变为两亲性的α-螺旋构象。Cathelicidin-BF的疏水侧插入到细菌细胞质膜内部，亲水侧暴露于细菌细胞质膜表面。随着结合到细菌细胞质膜上的cathelicidin-BF分子不断增加，细菌细胞质膜内陷，最终在细菌细胞质膜上形成孔洞。细菌细胞内容物大量外流，最终导致细菌细胞的死亡。 通过体外和体内多个实验对cathelicidin-BF进行了初步的药理学和药效学研究。Cathelicidin-BF在血清中稳定性较差，容易被血清中各种蛋白酶降解。一定浓度的盐离子能够增强cathelicidin-BF的抗菌活性。动物模型实验表明，cathelicidin-BF对多种细菌引起的小鼠皮肤感染具有很好的治疗效果。Cathelicidin-BF本身所具有的特点及动物模型实验中表现出的极佳的治疗效果使其成为外用抗菌药物开发的优良模板。

Cathelicidin LL-37:a defense molecule with a potential role in psoriasis pathogenesis

Epidermal keratinocytes produce and secrete antimicrobial peptides (AMPs) that subsequently form a chemical shield on the skin surface. Cathelicidins are one family of AMPs in skin with various further immune functions. Consequently, dysfunction of these peptides has been implicated in the pathogenesis of inflammatory skin disease. In particular, the cathelicidin LL-37 is overexpressed in inflamed skin in psoriasis, binds to extracellular self-DNA released from dying cells and converts self-DNA in a potent stimulus for plasmacytoid dendritic cells (pDCs). Subsequently, pDCs secrete type I interferons and trigger an auto-inflammatory cascade. Paradoxically, therapies targeting the vitamin D pathway such as vitamin D analogues or UVB phototherapy ameliorate cutaneous inflammation in psoriasis but strongly induce cathelicidin expression in skin at the same time. Current evidence now suggests that self-DNA present in the cytosol of keratinocytes is also pro-inflammatory active and triggers IL-1β secretion in psoriatic lesions through the AIM2 inflammasome. This time, however, binding of LL-37 to self-DNA neutralizes DNA-mediated inflammation. Hence, cathelicidin LL-37 shows contrasting roles in skin inflammation in psoriasis and might serve as a target for novel therapies for this chronic skin disease.

Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions

The proinflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1β is mediated by inflammasomes; however, the mechanisms triggering IL-1β processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-γ induced AIM2, and cytosolic DNA triggered the release of IL-1β via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1β activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.

Impact of vitamin D3 on cutaneous immunity and antimicrobial peptide expression

Antimicrobial peptides (AMPs) are effectors of cutaneous innate immunity and protect primarily against microbial infections. An array of AMPs can be found in and on the skin. Those include peptides that were first discovered for their antimicrobial properties but also proteins with antimicrobial activity first characterized for their activity as chemokines, enzymes, enzyme inhibitors and neuropeptides. Cathelicidins were among the first families of AMPs discovered in skin. They are now known to exert a dual role in innate immune defense: they have direct antimicrobial activity and will also initiate a host cellular response resulting in cytokine release, inflammation and angiogenesis. Altered cathelicidin expression and function was observed in several common inflammatory skin diseases such as atopic dermatitis, rosacea and psoriasis. Until recently the molecular mechanisms underlying cathelicidin regulation were not known. Lately, vitamin D3 was identified as the major regulator of cathelicidin expression and entered the spotlight as an immune modulator with impact on both, innate and adaptive immunity. Therapies targeting vitamin D3 signalling may provide novel approaches for the treatment of infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions through AMP regulation.

Control of cutaneous antimicrobial peptides by vitamin D3

Constant exposure to a wide variety of microbial pathogens represents a major challenge for our skin. Antimicrobial peptides (AMPs) are mediators of cutaneous innate immunity and protect primarily against microbial infections. Cathelicidins were among the first AMPs identified in human skin and recent evidence suggests that they exert a dual role in innate immune defense: At first, due to their antimicrobial activity they kill pathogens directly. In addition, these peptides initiate a potent host response to infection resulting in cytokine release, inflammation and a cellular response. Disturbed cathelicidin expression and function was observed in several common inflammatory skin diseases, such as psoriasis where cathelicidin peptide converts inert self-DNA and self-RNA into an autoimmune stimulus. In atopic dermatitis decreased levels of cathelicidin facilitating microbial superinfections have been discussed. Furthermore, abnormally processed cathelicidin peptides induce inflammation and a vascular response in rosacea. Until recently, the molecular mechanisms underlying cathelicidin regulation were unknown. Recently, the vitamin D3 pathway was identified as the major regulator of cathelicidin expression. Consequently, vitamin D3 entered the spotlight as an immune modulator with impact on both innate and adaptive immunity. Therapies targeting vitamin D3 signaling may provide new approaches for infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions.

Estudo das catelicidinas no plasma seminal de garanhões e das interleucinas na resposta inflamatória pós-iseminação artificial em éguas

Pós-graduação em Medicina Veterinária - FMVZ