Reflections on a systematic nomenclature for antimicrobial peptides from the skins of frogs of the family Ranidae

Frogs belonging to the extensive family Ranidae represent a valuable source of antimicrobial peptides with therapeutic potential but there is currently no consistent system of nomenclature to describe these peptides. Terminology based solely on species name does not reflect the evolutionary relationships existing between peptides encoded by orthologous and paralogous genes. On the basis of limited structural similarity, at least 14 well-established peptide families have been identified (brevinin-1, brevinin-2, esculentin-1, esculentin-2, japonicin-1, japonicin-2, nigrocin-2, palustrin-1, palustrin-2, ranacyclin, ranalexin, ranatuerin-1, ranatuerin-2, temporin). It is proposed that terms that are synonymous with these names should no longer be used. Orthologous peptides from different species may be characterized by the initial letter of that species, set in upper case, with paralogs belonging to the same peptide family being assigned letters set in lower case, e.g. brevinin-1Pa, brevinin-1Pb, etc. When two species begin with the same initial letter, two letters may be used, e.g. P for pipiens and PL for palustris. Species names and assignments to genera may be obtained from Amphibian Species of the World Electronic Database, accessible at http://research.amnh.org/herpetology/amphibia/index.php. American Museum of Natural History, New York, USA.     

蛙科两栖动物皮肤抗菌肽的分子多样性及功能

摘要: 蛙科(Ranidae)是全球分布最广泛的两栖动物, 种类超过650种。为开拓和适应广阔的栖息地及多样的生态环境, 其皮肤腺体中进化产生了结构复杂、种类繁多的抗菌肽。它们除具有广谱抗菌活性外, 还有抗肿瘤、抗病毒等生物学活性。蛙科动物皮肤抗菌肽起源于共同祖先, 在漫长的进化过程中, 基因发生了多重复制和突变, 形成了天然抗菌肽的巨大资源库。这些肽在模拟膜的溶剂中几乎都是疏水的, 并带正电荷, 以一种两亲性的α-螺旋的构象存在。根据氨基酸组成及结构的相似性, 可以将蛙科动物抗菌肽分成brevinin-1、esculentin-1、esculentin-2、temporin、ranalexin、ranatuerin-1、ranatuerin-2、plaustrin、brevinin-2、tigerinin、japonicin、nigrocin和melittin相关肽等若干个家族。文章结合作者的研究工作, 综述了目前已经鉴定的蛙科动物皮肤抗菌肽的分子多样性特点、家族性质和生物学活性的研究进展, 并阐述了东北林蛙新家族抗菌肽的特点。 关键词: 蛙科; 抗菌肽家族; 分子多样性; 生物学活性     

Active peptides in the skins of two hundred and thirty American amphibian species

Extracts prepared from dried or fresh skins of more than 200 American amphibian species were subjected to biological screening in order to determine occurrence and contents of peptides active on smooth muscle preparations, systemic blood pressure and, subordinately, external secretions, anterior pituitary and the central nervous system. The peptide families identified in skin extracts were as follows: caruleins (caerulein, phyllocaerulein), tachykinins (physalaemin, phyllomedusin), bombesins (phyllolitorin, [Leu8]phyllolitorin, rohdeilitorin), bradykinins (phyllokinin and others), sauvagine, dermorphins (dermorphin, [Hyp6]dermorphin), tryptophyllins (numerous peptides) and, finally, miscellaneous peptides. None of the above peptide families showed a widespread distribution, but all were restricted to particular amphibian genera or stocks. The hylid frogs of the Phyllomedusinae family occupy a unique position, as their skin displayed the greatest variety and abundance of active peptides ever found in any amphibian is destined to increase because numerous other peptide molecules await isolation, elucidation of structure and definition of possible biological activities.     

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.     

Parameters involved in antimicrobial and endotoxin detoxification activities of antimicrobial peptides

Endotoxin [lipopolysaccharide (LPS)] covers more than 90% of the outer monolayer of the outer membrane of Gram-negative bacteria, and it plays a dual role in its pathogenesis: as a protective barrier against antibiotics and as an effector molecule, which is recognized by and activates the innate immune system. The ability of host-defense antimicrobial peptides to bind LPS on intact bacteria and in suspension has been implicated in their antimicrobial and LPS detoxification activities. However, the mechanisms involved and the properties of the peptides that enable them to traverse the LPS barrier or to neutralize LPS endotoxic activity are not yet fully understood. Here we investigated a series of antimicrobial peptides and their analogues with drastically altered sequences and structures, all of which share the same amino acid composition (K 6L 9). The list includes both all- l-amino acid peptides and their diastereomers (composed of both l- and d-amino acids). The peptides were investigated functionally for their antibacterial activity and their ability to block LPS-dependent TNF-alpha secretion by macrophages. Fluorescence spectroscopy and transmission electron microscopy were used to detect their ability to bind LPS and to affect its oligomeric state. Their secondary structure was characterized in solution, in LPS suspension, and in LPS multibilayers by using CD and FTIR spectroscopy. Our data reveal specific biophysical properties of the peptides that are required to kill bacteria and/or to detoxify LPS. Besides shedding light on the mechanisms of these two important functions, the information gathered should assist in the development of AMPs with potent antimicrobial and LPS detoxification activities.     

Active peptides in the skins of one hundred amphibian species from Australia and Papua New Guinea

Extracts prepared from the dried skins of approximately one hundred amphibian species from Australia and Papua New Guinea were subjected to biological screening in order to determine the nature and amounts of peptides active on smooth muscle preparations and systemic blood pressure present in these extracts. The most frequently and abundantly occurring peptides were those of the caerulein, bombesin and tachykinin peptide families represented, respectively, by caerulein; litorin, Glu(OMe)2-litorin and Glu(OEt)2-litorin; uperolein and Lys5-Thr6-physalaemin. Bradykinin-like peptides seem to have a rather diffuse distribution, in the species examined, but so far no peptide of this family has been isolated and sequenced. The only angiotensin-like peptide ever found in amphibian skin, crinia angiotensin II, has been isolated from skin extracts of a few species, belonging to the genera Crinia, Geocrinia, Ranidella and Litoria. The array of peptides occurring in amphibians from Australia and Papua New Guinea is destined to increase, because several apparently novel peptides have been identified in skin extracts by bioassay and radioimmunoassay.     

A tridecapeptide possesses both antimicrobial and protease-inhibitory activities

A 13-residue synthetic peptide (Rev4) was designed based on indolicidin, an antimicrobial peptide from bovine neutrophils. The synthetic peptide retains high antimicrobial activity. When tested for its stability in tobacco leaf extracts, Rev4 was highly stable compared to another antimicrobial peptide, magainin. When mixed with Rev4, magainin was protected from degradation by the leaf extract. Our results show that Rev4 is a potent protease inhibitor which selectively inhibits three out of four different types of proteases. Four other synthetic peptides were tested and the results were suggestive of no correlation between their antimicrobial and protease inhibitory activities.     

The role of amphibian antimicrobial peptides in protection of amphibians from pathogens linked to global amphibian declines

Amphibian species have experienced population declines and extinctions worldwide that are unprecedented in recent history. Many of these recent declines have been linked to a pathogenic skin fungus, Batrachochytrium dendrobatidis, or to iridoviruses of the genus Ranavirus. One of the first lines of defense against pathogens that enter by way of the skin are antimicrobial peptides synthesized and stored in dermal granular glands and secreted into the mucus following alarm or injury. Here, I review what is known about the capacity of amphibian antimicrobial peptides from diverse amphibians to inhibit B. dendrobatidis or ranavirus infections. When multiple species were compared for the effectiveness of their in vitro antimicrobial peptides defenses against B. dendrobatidis, non-declining species of rainforest amphibians had more effective antimicrobial peptides than species in the same habitat that had recently experienced population declines. Further, there was a significant correlation between the effectiveness of the antimicrobial peptides and resistance of the species to experimental infection. These studies support the hypothesis that antimicrobial peptides are an important component of innate defenses against B. dendrobatidis. Some amphibian antimicrobial peptides inhibit ranavirus infections and infection of human T lymphocytes by the human immunodeficiency virus (HIV). An effective antimicrobial peptide defense against skin pathogens appears to depend on a diverse array of genes expressing antimicrobial peptides. The production of antimicrobial peptides may be regulated by signals from the pathogens. However, this defense must also accommodate potentially beneficial microbes on the skin that compete or inhibit growth of the pathogens. How this delicate balancing act is accomplished is an important area of future research.     

Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae)

As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC-MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.     

Extremely abundant antimicrobial peptides existed in the skins of nine kinds of Chinese odorous frogs

Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structure-functional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics.     

Virus infections in wild plant populations are both frequent and often unapparent

? Premise of the study: Pathogens are thought to regulate host populations. In agricultural crops, virus infection reduces yield. However, in wild plants little is known about the spatial and temporal patterns of virus prevalence. Thus, pathogen effects on plant population dynamics are unclear. Prevalence data provide necessary background for (1) evaluating the effects of virus infection on plant population size and dynamics and (2) improving risk assessment of virus-resistant transgenic crops. ? Methods: We used ELISA and RT-PCR to survey wild Cucurbita pepo populations over 4 years for five viruses, aphid-transmitted viruses of the genus Potyvirus as a group and PCR to survey for virus-resistance transgenes. In addition, we surveyed the literature for reports of virus prevalence in wild populations. ? Key results: In 21 C. pepo populations, virus prevalence (0-74%) varied greatly among populations, years, and virus species. In samples analyzed by both ELISA and RT-PCR, RT-PCR detected 6-44% more viruses than did ELISA. Eighty percent of these infections did not cause any visually apparent symptoms. In our samples, the virus-resistance transgene was not present. In 30 published studies, 92 of 146 tested species were infected with virus, and infection rates ranged from 0.01-100%. Most published studies used ELISA, suggesting virus prevalence is higher than reported. ? Conclusions: In wild C. pepo, the demographic effects of virus are likely highly variable in space and time. Further, our literature survey suggests that such variation is probably common across plant species. Our results indicate that risk assessments for virus-resistant transgenic crops should not rely on visual symptoms or ELISA and should include data from multiple populations over multiple years.     

Structural features and biological activities of the cathelicidin-derived antimicrobial peptides

Cathelicidins are a numerous group of mammalian proteins that carry diverse antimicrobial peptides at the C-terminus of a highly conserved preproregion. These peptides, which become active when released from the proregion, display a remarkable variety of sizes, sequences, and structures, and in fact comprise representatives of all the structural groups in which the known antimicrobial peptides have been classified. Most of the cathelicidin-derived peptides exert a broad spectrum and potent antimicrobial activity and also bind to lipopolysaccharide and neutralize its effects. In addition, some of them have recently been shown to exert other activities and might participate in host defense also by virtue of their ability to induce expression of molecules involved in a variety of biological processes. This review is aimed at providing a general overview of the cathelicidins and of the peptides derived therefrom, with emphasis on aspects such as structure, biological activities in vitro and in vivo, and structure/activity relationship studies.     

Role of membranes in the activities of antimicrobial cationic peptides

Cationic amphiphilic peptides that are found throughout nature have very broad-spectrum activities against microbes. The initial sites of interaction are with microbial membranes. Although dogma suggests that their lethal action involves disruption of the cytoplasmic membranes, a number of cationic peptides can traverse intact membranes to interact with internal targets.     

Synthesis of eperezolid-like molecules and evaluation of their antimicrobial activities

3-Fluoro-4-(4-phenylpiperazin-l-yl)aniline (II) prepared from 3,4-difluoro nitrobenzene was converted to the corresponding Schiff bases (III) and (IV) by treatment with 4-methoxybenzaldehyde and indol-3-carbaldehyde, respectively. Treatment of amine (II) with 4-fluorophenyl isothiocyanate afforded the corresponding thiourea derivative (V). Compound (V) was converted to thiazolidinone and thiazoline derivatives (VI) and (VII) by cyclocondensation with ethylbromoacetate or 4-chlorophenacylbromide, respectively. The synthesis of carbothioamide derivative (X) was performed starting from compound (II) by three steps. Treatment of compound (X) with sodium hydroxide, sulfuric acid, or chlorophenacyl bromide generated the corresponding 1,2,4-triazole (XI), 1,3,4-thiadiazole (XII), and 1,3-thiazolidinone (XIII) derivatives, respectively. The structural assignments of new compounds were based on their elemental analysis and spectral (IR, ¹H-NMR, ¹³C-NMR, and LC-MS) data. In the antimicrobial activity study all the compounds revealed high anti-Mycobacterium smegmatis activity.     

Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application

New peptides for lipopolysaccharide (LPS) and lipoteichoic acid (LTA) neutralization in upper respiratory tract infections were developed and evaluated in terms of efficacy and safety for application in humans. Based on the sequence of the human antimicrobial peptide LL-37 we developed and investigated length variants, substitution analogues and modifications to stabilize the peptides to prevent enzymatic degradation and to improve efficacy. The most promising peptide appears P60.4, a 24 amino acid peptide with similar efficacy as LL-37 in terms of LPS and LTA neutralization and lower pro-inflammatory activity. In addition, the acetylated and amidated version of this peptide shows no toxicity and displays higher or equal antimicrobial activity compared to LL-37.     

Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.     

Plant natriuretic peptides are apoplastic and paracrine stress response molecules

Higher plants contain biologically active proteins that are recognized by antibodies against human atrial natriuretic peptide (ANP). We identified and isolated two Arabidopsis thaliana immunoreactive plant natriuretic peptide (PNP)-encoding genes, AtPNP-A and AtPNP-B, which are distantly related members of the expansin superfamily and have a role in the regulation of homeostasis in abiotic and biotic stresses, and have shown that AtPNP-A modulates the effects of ABA on stomata. Arabidopsis PNP (PNP-A) is mainly expressed in leaf mesophyll cells, and in protoplast assays we demonstrate that it is secreted using AtPNP-A:green fluorescent protein (GFP) reporter constructs and flow cytometry. Transient reporter assays provide evidence that AtPNP-A expression is enhanced by heat, osmotica and salt, and that AtPNP-A itself can enhance its own expression, thereby generating a response signature diagnostic for paracrine action and potentially also autocrine effects. Expression of native AtPNP-A is enhanced by osmotica and transiently by salt. Although AtPNP-A expression is induced by salt and osmotica, ABA does not significantly modulate AtPNP-A levels nor does recombinant AtPNP-A affect reporter expression of the ABA-responsive RD29A gene. Together, these results provide experimental evidence that AtPNP-A is stress responsive, secreted into the apoplastic space and can enhance its own expression. Furthermore, our findings support the idea that AtPNP-A, together with ABA, is an important component in complex plant stress responses and that, much like in animals, peptide signaling molecules can create diverse and modular signals essential for growth, development and defense under rapidly changing environmental conditions.