Sanil George

Novel antibacterial peptides from the skin secretion of the Indian bicoloured frog Clinotarsus curtipes.

HPLC elution profile and MALDI TOF MS analysis of electro-stimulated skin secretion of the Indian Ranid frog Clinotarsus curtipes of the Western Ghats confirmed the presence of multiple peptides. Peptides eluted out of the C18 column at higher hydrophobic solvent region showed antibacterial activity against diverse bacterial strains, including the clinical isolates of V. cholerae and methicillin resistant Staphylococcus aureus (MRSA). Peptidomic analysis of the most potent chromatographic effluent fraction identified five novel peptide amides having sequence homology with brevinin family. These peptides are named as brevinin1CTcu1 (B1CTcu1) to brevinin1CTcu5 (B1CTcu5). Peptide B1CTcu1 is non-haemolytic while the others are haemolytic in nature but all elicited potential antibacterial activity. B1CTcu5 is a twenty-one residue peptide amide having proline hinge region in the middle and the typical C-terminal intramolecular disulfide-bridged hepta peptide domain (Rana box) that is present in most of the brevinin peptides. Analysis of their killing kinetics with E. coli and S. aureus and the ability to induce membrane depolarization proved that these are two independent events. These novel multifunctional peptides play an important role to protect C. curtipes from invading pathogenic microorganisms present in the environment.

A mini review on the antimicrobial peptides isolated from the genus Hylarana (Amphibia: Anura) with a proposed nomenclature for amphibian skin peptides

Hylarana is a well established frog genus coming under the family Ranidae. An increasing number of antimicrobial peptides have been isolated and characterized from the skin of frogs of this genus. This review covers the antimicrobial peptides reported so far from the frogs of Hylarana genus and to propose a consistent system of nomenclature for amphibian skin peptides. Multiple sequence alignment of the skin peptides from Hylarana genus has grouped them into six peptide families, and three bioactive peptides. Existing nomenclature of amphibian antimicrobial peptides is species centered with no implication to the genus which can lead to disparities, when frogs with same species name belonging to different genus have to be named. As per the proposed system the peptide should have the parent peptide name (e.g. Brevinin-1) followed by two uppercase letter of the genus, if two genera begin with the same letter–first letter should be the same followed by an appropriate second letter (e.g. HU for Huia and HM for Humenerana). This is succeeded by species name in lower case-orthologous peptides from different species may be characterized by the initial letter of that species, when two species begin with the same initial letter, second letter should be used appropriately (e.g. HLat for Hylarana aurata and HLan for Hylarana aurantiaca). Paralogs belonging to the same peptide family are assigned by numbers.

Endemic Indirana Frogs of the Western Ghats Biodiversity Hotspot

Frogs of the genus Indirana belong to the endemic family Ranixalidae and are found exclusively in the Western Ghats biodiversity hotspot. Since taxonomy, biology and distribution of these frogs are still poorly understood, we conducted a comprehensive literature review of what is known on the taxonomy, morphology, life history characteristics and breeding biology of these species. Furthermore, we collected information on the geographical locations mentioned in the literature, and combined this with information from our own field surveys in order to generate detailed distribution maps for each species. Apart from serving as a useful resource for future research and conservation efforts, this review also highlights the areas where future research efforts should be focussed.

Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides

All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.

Cross-species testing and utility of microsatellite loci in Indirana frogs

BackgroundMicrosatellite loci are widely used in population and conservation genetic studies of amphibians, but the availability of such markers for tropical and subtropical taxa is currently very limited. In order to develop resources for conservation genetic studies in the genus Indirana, we tested amplification success and polymorphism in 62 previously developed microsatellite loci, in eight Indirana species - including new candidate species. Developing genomic resources for this amphibian taxon is particularly important as it is endemic to the Western Ghats biodiversity hotspot, and harbours several endangered species.FindingsThe cross-species amplification success rate varied from 11.3 % to 29.0 % depending on the species, with 29 - 80 % of the amplifying loci being polymorphic. A strong negative correlation between cross-species amplification success (and polymorphism) and genetic distance separating target from source species was observed.ConclusionsOur results provide additional genetic support for the existence of genetically divergent cryptic species within the genus Indirana. The tested markers should be useful for population and conservation genetic studies in this genus, and in particular, for species closely related to the source species, I. beddomii.

Identification and characterization of novel host defense peptides from the skin secretion of the fungoid frog, Hydrophylax bahuvistara (Anura: Ranidae)

Two novel peptides (brevinin1 HYba1 and brevinin1 HYba2) were identified from the skin secretion of the frog Hydrophylax bahuvistara, endemic to Western Ghats, India, and their amino acid sequences were confirmed using cDNA cloning and LC/MS/MS. Antibacterial, hemolytic, and cytotoxic activities of brevinin1 peptides and their synthetic analogs (amidated C‐terminus) were investigated and compared. All the peptides except the acidic forms showed antibacterial activity against all tested Gram‐positive and Gram‐negative bacteria. They exhibited low hemolysis on human erythrocytes and showed potent cytotoxic activity against Hep 3B cancer cell line. Upon amidation, the peptides showed increased activity against the tested microbes without altering their hemolytic and cytotoxic properties. The study also emphasizes the need for screening endemic amphibian fauna of Western Ghats, as a potential source of host defense peptides with possible therapeutic applications in the future.

The status of Eurostus dussumierii and Hypsirhina chinensis (Reptilia, Squamata, Serpentes): with comments on the origin of salt tolerance in homalopsid snakes

The Kerala mud snake, Enhydris dussumierii Duméril, Bibron & Duméril, has long been known only from syntypes collected in the nineteenth century, but more recent specimens have provided the opportunity for molecular work. Using Bayesian phylogenetic analysis of 2200 base pairs of cytb, 16S, and c-mos, we recovered the Kerala mud snake as the sister species to the Chinese mud snake, Enhydris chinensis (Chinese–Vietnamese endemic). The DNA results establish the position of dussumierii and chinensis as separate from the Enhydris clade, and together they form the sister group to most of the other fanged homalopsids ((Bitia+Cantoria) + (Erpeton+Gerarda+Fordonia) + (Myron+Pseudoferania) + (Enhydris punctata) + (Homalopsis+Enhydris bocourti) + (Cerberus)). Here we resurrect Ferania Gray for Enhydris sieboldii Gray, establish a new genus for the E. chinensis clade (bennettii+chinensis), and apply the generic replacement name Dieurostus Berg to E. dussumierii Duméril, Bibron & Duméril. A biogeographic scenario is proposed based on the ecology, distribution and salt tolerance of the species composing a hypothesized (not supported with molecular evidence) Asian coastal lineage that is distributed from Pakistans Indus River delta to temperate coastal China.

Use of DNA barcoding in wildlife forensics: a study of sambar deer (Rusa unicolor)

This paper reports a case in which a wildlife range officer had suspected that some people had killed a sambar deer (Rusa unicolor). Cooked meat and dried skin were seized from the suspects and forwarded to our laboratory for DNA testing to resolve the identity of the animal. Mitochondrial cytochrome C oxidase subunit 1 (CO1) analysis revealed that the cooked meat and skin of the animal were of a sambar deer by comparing the sequences with the reference sequence of R. unicolor generated in our laboratory. The study illuminates the use of barcode sequences and reference databases in wildlife forensic applications.

Studies on the Mode of Membrane Interaction of C-terminally Amidated Brevinin1 HYba1 and 2 Peptides Against Bacteria

In this study, we investigated the possible mode of action of two C-terminally amidated novel peptides identified from the frog skin on both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio cholerae). The results indicate that the peptides initially induce membrane depolarization followed by pore formation in a concentration-dependent manner. A microscopic examination revealed the fact that the peptides are capable of destroying bacterial cells physically. The activity of these peptides against Gram-negative bacteria was dependent on the presence of divalent cations (Ca2+ and Mg2+) but not in Gram-positive bacteria. This study also emphasizes that the endemic frogs of the Western Ghats may provide a valuable source of its skin peptides having the potential for further improvement as lead drug molecules.

First report of Lividin and Spinulosain peptides from the skin secretion of an Indian frog

Here, we report two novel peptides identified from the skin secretion, having homologies to Lividin and Spinulosain, of an endemic frog, Hydrophylax bahuvistara, of Western Ghats. This is the first report of these peptides from Indian frogs and first identification of Lividin from the Hydrophylax genus. Both peptides exhibited weak antimicrobial activity but very low haemolytic activity. The problems of naming amphibian host defense peptides (HDPs) are also discussed.