Identification and Characterization of the First Cathelicidin from Sea Snakes with Potent Antimicrobial and Anti-inflammatory Activity and Special Mechanism

Cathelicidins are a family of gene-encoded peptide effectors of innate immunity found exclusively in vertebrates. They play pivotal roles in host immune defense against microbial invasions. Dozens of cathelicidins have been identified from several vertebrate species. However, no cathelicidin from marine reptiles has been characterized previously. Here we report the identification and characterization of a novel cathelicidin (Hc-CATH) from the sea snake Hydrophis cyanocinctus. Hc-CATH is composed of 30 amino acids, and the sequence is KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL. Circular dichroism spectroscopy and structure modeling analysis indicated that Hc-CATH mainly assumes an amphipathic α-helical conformation in bacterial membrane-mimetic solutions. It possesses potent broad-spectrum and rapid antimicrobial activity. Meanwhile, it is highly stable and shows low cytotoxicity toward mammalian cells. The microbial killing activity of Hc-CATH is executed through the disruption of cell membrane and lysis of bacterial cells. In addition, Hc-CATH exhibited potent anti-inflammatory activity by inhibiting the LPS-induced production of nitric oxide (NO) and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Hc-CATH directly binds with LPS to neutralize its toxicity, and it also binds to Toll-like receptor 4 (TLR4/MD2 complex), which therefore inhibits the binding of LPS to TLR4/MD2 complex and the subsequent activation of LPS-induced inflammatory response pathways. Taken together, our study demonstrates that Hc-CATH, the first cathelicidin from sea snake discovered to have both antimicrobial and anti-inflammatory activity, is a potent candidate for the development of peptide antibiotics.     

抗海蛇毒血清用于海蛇咬伤早期急救与监护的实验研究

目的观察抗青环海蛇毒血清用于海蛇咬伤动物模型早期急救的效果及监护重点的筛选,为抗海蛇毒血清的临床应用提供实验依据。方法将40只新西兰兔随机分成5组,采用大腿肌肉注射青环海蛇毒素拟海蛇咬伤模型,分别于中毒后0.5h、2h及4h给予抗海蛇毒血清及相应的急救措施,连续监测各项生理和生化指标以及24h存活情况。结果0.5h和2h给予抗海蛇毒血清组动物的各项指标明显优于4h应用抗海蛇毒血清组,24h存活情况良好;4h应用抗海蛇毒血清组中,早期进行呼吸支持组的部分指标优于无呼吸支持组,24h存活率提高。结论抗青环海蛇毒血清是有效的对抗海蛇咬伤的急救药物,越早使用效果越好,对各脏器能起到较好的保护作用。在丧失最佳应用抗海蛇毒血清的情况下,尽早应用呼吸机进行人工呼吸支持,有助于提高后期的存活率,减少发展成危重病例的可能,为进一步抢救赢得时机。     

Snake venomics of two poorly known Hydrophiinae: Comparative proteomics of the venoms of terrestrial Toxicocalamus longissimus and marine Hydrophis cyanocinctus

The venom proteomes of Toxicocalamus longissimus and Hydrophis cyanocinctus, a fossorial and a marine species, respectively, of the Hydrophiinae genus of Elapidae, were investigated by Edman degradation of RP-HPLC isolated proteins, and de novo MS/MS sequencing of in-gel derived tryptic peptide ions. The toxin arsenal of T. longissimus is made up of 1-2 type-I PLA(2) molecules, which account for 6.5% of the venom proteins, a minor PIII-SVMP (1.4% of the venom toxins), and ~20 members of the 3FTx family comprising 92% of the venom proteome. Seventeen proteins (5 type-I PLA(2)s and 12 3FTxs) were found in the venom of H. cyanocinctus. Three-finger toxins and type-I PLA(2) proteins comprise, respectively, 81% and 19% of its venom proteome. The simplicity of the H. cyanocinctus venom proteome is highlighted by the fact that only 6 venom components (3 short-chain neurotoxins, two long-chain neurotoxins, and one PLA(2) molecule) exhibit relative abundances >5%. As expected from its high neurotoxin abundance, the LD(50) for mice of H. cyanocinctus venom was fairly low, 0.132μg/g (intravenous) and 0.172μg/g (intraperitoneal). Our data indicate that specialization towards a lethal cocktail of 3FTx and type-I PLA(2) molecules may represent a widely adopted trophic solution throughout the evolution of Elapidae. Our results also points to a minimization of the molecular diversity of the toxin arsenal of the marine snake Hydrophis cyanocinctus in comparison to the venom proteome of its terrestrial relatives, and highlight that the same evolutionary solution, economy of the toxin arsenal, has been convergently adopted by different taxa in response to opposite selective pressures, loss and gain of neurotoxicity.     

Review of sea snakes of the genus <Emphasis Type="Italic">Hydrophis</Emphasis> sensu stricto (Serpentes: Hydrophiidae)

The results of the genus Hydrophis revision s. str. are presented. The genus is divided into three subgenera, including Mediohydrophis subgen. nov. The validity of Hydrophis atriceps and H. (Microcephalophis) gracilis microcephalus is confirmed. New data on the distribution of H. klossi is given.Original Russian Text Copyright © 2004 by Biologiya Morya, Kharin.