A novel anionic cathelicidin lacking direct antimicrobial activity but with potent anti-inflammatory and wound healing activities from the salamander Tylototriton kweichowensis.

Cathelicidin is a family of antimicrobial peptides (AMPs) existing in vertebrates, which play multiple functions in host responses against environmental stresses. All cathelicidins identified to date are cationic, no anionic member with net negative charges has been reported. In the present study, a novel anionic cathelicidin (TK-CATH) with a net charge of -3 was identified from the skin of the salamander, T. kweichowensis. Unlike most other cathelicidin members, it didn't exhibit direct antimicrobial activity. However, it demonstrated strong anti-inflammatory activity. It effectively inhibited the LPS-induced pro-inflammatory cytokine gene expression and protein production in amphibian leukocytes and mouse macrophages by inhibiting the LPS-activated mitogen-activated protein kinase (MAPK) signaling pathways. Besides, TK-CATH showed potent wound healing activity. It could effectively induce the production of several cytokines, chemokines and growth factors relating to wound healing, promote the motility and proliferation of keratinocytes, and accelerate the skin wound healing in a mouse full-thickness wound model. These results imply that TK-CATH participates in both the inflammatory phase and new tissue formation phase of wound repair process. Meanwhile, TK-CATH exhibited weak but effective free radical scavenging activity and low cytotoxicity. All the results above indicate that TK-CATH is a multifunctional peptide in the skin of the salamander T. kweichowensis. It may play important roles in host immune responses against bacterial infection and skin wound repair.

Identification and characterization of novel bi-functional cathelicidins from the black-spotted frog (Pelophylax nigromaculata) with both anti-infective and antioxidant activities.

Cathelicidins are an important family of antimicrobial peptides (AMPs), which play pivotal roles in vertebrate immune responses against microbial infections. They are regarded as potential drug leads for the development of novel antimicrobial agents and three related drugs have been developed into clinical trials. Thus, it is meaningful to identify more cathelicidins from vertebrate species. Cathelicidins from ranid frogs possess special structural characteristics and activities, but to date only 12 ranid frog cathelicidins have been identified. In the present study, two novel cathelicidins (PN-CATH1 and 2) were identified from the black-spotted frog, Pelophylax nigromaculata. PN-CATHs possess low sequence similarity with the known cathelicidins. They exhibited moderate, but broad-spectrum and rapid antimicrobial activities against the tested bacteria. They kill bacteria by mainly inducing bacterial membrane disruption and possibly generating intracellular ROS formation. They also possess potent anti-biofilm and persister cell killing activity, indicating their potential in combating infections induced by biofilms-forming bacteria. Besides direct antimicrobial activity, they exhibited potent anti-inflammatory activity by effectively inhibiting the LPS-induced production of pro-inflammatory cytokines in mouse macrophages, which could be partly ascribed to their direct LPS-neutralizing ability. Furthermore, PN-CATHs demonstrated powerful in vitro free radical scavenging activities. Ultraviolet radiation significantly increased their in vivo gene expression in frog skin. Meanwhile, they possess weak cytotoxic activity and extremely low hemolytic activity. PN-CATHs represent the first discovery of cathelicidins family AMPs with both potent anti-infective and antioxidant activities. The discovery of PN-CATHs provides potential peptide leads for the development of novel anti-infective and antioxidant drugs.

Design of a Sea Snake Antimicrobial Peptide Derivative with Therapeutic Potential against Drug-Resistant Bacterial Infection.

Infections caused by drug-resistant pathogens are a worldwide challenge for public health. Antimicrobial peptides (AMPs) are regarded as promising antibiotic alternatives for the treatment of drug-resistant infections. In the present study, a series of small peptides were designed based on our previously reported sea snake AMP Hc-CATH. From them, the lead peptide HC1-D2, a truncated peptide entirely substituted by d-amino acids, was selected. HC1-D2 exhibited significantly improved stability and antibiofilm and anti-inflammatory activities. Meanwhile, HC1-D2 retained potent, broad-spectrum, and rapid antimicrobial properties against bacteria and fungi, especially drug-resistant bacteria. Moreover, HC1-D2 showed low propensity to induce bacterial resistance and low cytotoxicity and hemolytic activity. Notably, HC1-D2 showed potent in vivo anti-infective ability in mouse peritonitis models infected by both standard and drug-resistant bacteria. It significantly decreased the bacterial counts in the abdominal cavity and spleen of mice and apparently increased the survival rates of the mice. Acting through the MAPKs inflammatory pathway, HC1-D2 selectively induced the production of chemokine and the subsequent immune cell recruitment to the infection site, while inhibiting the production of pro-inflammatory cytokines with undesirable toxicities. These much improved properties make HC1-D2 a promising candidate for the development of novel peptide anti-infective agents against drug-resistant infections.