A Novel Brevinin2 HYba5 Peptide against Polymicrobial Biofilm of Staphylococcus aureus and Enterococcus faecalis.

BACKGROUND: Brevinin2 HYba5 (Peptide 29) is a novel cationic peptide identified from an endemic frog, Hydrophylax bahuvistara. Staphylococcus aureus and Enterococcus faecalis are troublesome biofilm-forming pathogens associated with nosocomial and community-acquired infections and contribute to the severity of infections associated with implanted devices and chronic wounds. Co-existence of both pathogens in biofilm mode contributes to an increased antibiotic resistance, treatment failure and hence persistent disease burden. Identifying a novel and stable, less toxic compound targeting multispecies biofilm with a lower probability of acquiring resistance in comparison to antibiotics is highly warranted. OBJECTIVE: Evaluate the activity of Brevinin2 HYba5 against S. aureus and E. faecalis mixed biofilm. METHODS: The anti-biofilm activity of peptide 29 was tested by Crystal violet assay, Confocal laser scanning Microscopy (CLSM) and MTT Assay. Cytotoxicity of the peptide was tested in RBC and L929 fibroblast cell line. Biofilm inhibitory activity of the peptide was evaluated at different temperatures, pH, serum and plasma concentrations. The antibiofilm potential of the peptide was tested against polymicrobial biofilm by Fluorescent in situ hybridisation (FISH) and plate counting on HiCromeTM UTI Agar media. RESULTS: The peptide 29 could inhibit biofilm formation of S. aureus and E. faecalis individually as well as in polymicrobial biofilm at 75 µM concentration. The peptide maintained its antibiofilm potential at different temperatures, serum and plasma concentrations. Activity of the peptide was high at acidic and neutral pH but found to get reduced towards alkaline pH. The peptide is nonhemolytic and does not exhibit significant cytotoxicity against the L929 fibroblast cell line (92.80% cell viability). CONCLUSION: The biofilm inhibition property makes peptide 29 a promising candidate for the management of S. aureus and E. faecalis biofilm, especially in catheter-associated devices to prevent the initial colonization and thus can ease the burden of pathogenic biofilm-associated infections.

Population genetic structure and evolutionary demographic patterns of Phrynoderma karaavali, an edible frog species of Kerala, India.

Trade and collection of edible frogs are banned in India. We used mitochondrial (16 and 12S DNA) and nuclear gene (Rag-1 and Rhodopsin) sequences to examine the population genetic and demographic structure of an edible frog species, Phrynoderma karaavali (Karaavali Skittering frog) from Kerala as it exist after the ban. Frogs from 11 sites show high mtDNA haplotype and nDNA diversity which indicates a stable or expanding population. The evolutionary demographic pattern suggests population expansion across its geographical range, even though the species is still subject to poaching. Two major population clusters were observed at the northern and southern end of the species range. Gene flow occurs despite of geographic barriers. Genetic distance increases with geographical distance. P. karaavali diverged from its sister species in Phrynoderma around 11 mya in the late Miocene.

The complete mitochondrial genome of an endemic cichlid Etroplus canarensis from Western Ghats, India (Perciformes: Cichlidae) and molecular phylogenetic analysis.

BACKGROUND: The Indian endemic cichlid Etroplus canarensis (Canara pearl spot) is an endangered fish and is one among the three Indian cichlids (Etroplinae) that had a restricted distribution in the South Canara region of Karnataka, India. Despite considerable investigations, the phylogeny of Indian Cichlids and its biogeographical origin is still ambiguous and remains a question under discussion which is scrutinized based on whole mitogenomes in the present study. METHODS AND RESULTS: We report the 16,339 bp complete mitochondrial genome of E. canarensis for the first time using the next-generation sequencing methods. Comparison of gene arrangement and genome characterization was found to commensurate with the previous reports on two Indian cichlid fishes, E. suratensis and E. maculatus. ND6 has been identified as a gene with the highest evolutionary rate and COI and COII is the most conserved gene based on p-genetic distance calculation. Substitution rate (ka/ks) was found to be very low indicating a reduced rate of evolution among subfamily Etroplinae accounting for its subsided species divergence of Indian cichlids. Phylogenetic analysis of Indian cichlids based on a combined dataset of 12 protein-coding genes representing cichlids generated high posterior probability values pillaring paraphyletic nature of Indian Malagasy lineage and monophyletic Indian genus Etroplus. CONCLUSION: The mitogenome sequence of E. canarensis may provide fundamental molecular data useful for further researches on genetic diversity, endemicity and the conservation of this endangered freshwater fish.

SSTP1, a Host Defense Peptide, Exploits the Immunomodulatory IL6 Pathway to Induce Apoptosis in Cancer Cells.

While the immunomodulatory pathways initiated in immune cells contribute to therapeutic response, their activation in cancer cells play a role in cancer progression. Also, many of the aberrantly expressed immunomodulators on cancer cells are considered as therapeutic targets. Here, we introduce host defense peptide (HDP), a known immuomodulator, as a therapeutic agent to target them. The cationic host defense peptides (HDPs), an integral part of the innate immune system, possess membranolytic activity, which imparts antimicrobial and antitumor efficacy to it. They act as immunomodulators by activating the immune cells. Though their antimicrobial function has been recently reassigned to immunoregulation, their antitumor activity is still attributed to its membranolytic activity. This membrane pore formation ability, which is proportional to the concentration of the peptide, also leads to side effects like hemolysis, limiting their therapeutic application. So, despite the identification of a variety of anticancer HDPs, their clinical utility is limited. Though HDPs are shown to exert the immunomodulatory activity through specific membrane targets on immune cells, their targets on cancer cells are unknown. We show that SSTP1, a novel HDP identified by shotgun cloning, binds to the active IL6/IL6Rα/gp130 complex on cancer cells, rearranging the active site residues. In contrast to the IL6 blockers inhibiting JAK/STAT activity, SSTP1 shifts the proliferative IL6/JAK/STAT signaling to the apoptotic IL6/JNK/AP1 pathway. In IL6Rα-overexpressing cancer cells, SSTP1 induces apoptosis at low concentration through JNK pathway, without causing significant membrane disruption. We highlight the importance of immunomodulatory pathways in cancer apoptosis, apart from its established role in immune cell regulation and cancer cell proliferation. Our study suggests that identification of the membrane targets for the promising anticancer HDPs might lead to the identification of new drugs for targeted therapy.

The amphibian peptide Yodha is virucidal for Zika and dengue viruses.

Zika virus (ZIKV) has emerged as a serious health threat in the Americas and the Caribbean. ZIKV is transmitted by the bite of an infected mosquito, sexual contact, and blood transfusion. ZIKV can also be transmitted to the developing fetus in utero, in some cases resulting in spontaneous abortion, fetal brain abnormalities, and microcephaly. In adults, ZIKV infection has been correlated with Guillain-Barre syndrome. Despite the public health threat posed by ZIKV, neither a vaccine nor antiviral drugs for use in humans are currently available. We have identified an amphibian host defense peptide, Yodha, which has potent virucidal activity against ZIKV. It acts directly on the virus and destroys Zika virus particles within 5 min of exposure. The Yodha peptide was effective against the Asian, African, and South American Zika virus strains and has the potential to be developed as an antiviral therapeutic in the fight against Zika virus. The peptide was also effective against all four dengue virus serotypes. Thus, Yodha peptide could potentially be developed as a pan-therapeutic for Zika and dengue viruses.

Insights into Peptide Mediated Antibiofilm Treatment in Chronic Wound: A Bench to Bedside Approach.

Chronic wound biofilm infections are a threat to the population with respect to morbidity and mortality. The presence of multidrug-resistant bacterial pathogens in chronic wound renders the action of antibiotics and antibiofilm agents difficult. Therefore an alternative therapy is essential for reducing bacterial biofilm burden. In this scenario, the peptide-based antibiofilm therapy for chronic wound biofilm management seeks more attention. A synthetic peptide with a broad range of antibiofilm activity against preformed and established biofilms, having the ability to kill multispecies bacteria within biofilms and possessing combinatorial activity with other antimicrobial agents, provides significant insights. In this review, we portray the possibilities and difficulties of peptide-mediated treatment in chronic wounds biofilm management and how it can be clinically translated into a product.

Investigations on the membrane interaction of C-terminally amidated esculentin-2 HYba1 and 2 peptides against bacteria.

The membrane interaction and damage caused by C-terminally amidated esculentin-2 peptides identified from the frog skin is illustrated in the present study using Staphylococcus aureus and Vibrio cholerae. Double staining with fluorescent probes SYTOX and DAPI proved the concentration-dependent bacterial membrane damage induced by the peptides. It was found that the sub-MIC of both peptides induced transient pores on the bacterial membrane. These peptides also caused depolarisation on the bacterial membrane during their interaction. The physical changes on bacterial cells like blebbing, elongation, fusion, and so forth upon peptide treatment were visualized through SEM images. The antimicrobial activity of the peptides against S. aureus and V. cholerae was not altered at physiological concentrations of divalent and monovalent cations, which is advantageous in a therapeutic context. The increase of MIC against V. cholerae at higher concentrations of Mg2+ and Ca2+ (>5 µM) is due to the concentration-dependent antagonism exhibited by these ions for the cation binding sites on the bacterial membrane, which facilitates the process of 'self-promoted uptake.' The study emphasizes to utilize the ability of these peptides to produce transient pores at sub-MICs in combinatorial therapy.

Identification and functional characterisation of Esculentin-2 HYba peptides and their C-terminally amidated analogs from the skin secretion of an endemic frog.

Here, we report the identification, functional characterisation, and the effect of C-terminal amidation on the activity profile of two novel Esculentin-2 peptides (Esculentin-2 HYba1 and Esculentin-2 HYba2). The parent peptides and their analogs exhibited potent activity against the tested Gram-positive and Gram-negative bacteria. The effect of amidation was evident in the activity profile of fish pathogens and killing kinetics. The analogs showed a 10-fold decrease in MIC, and the killing time was reduced to 10-15 minutes. The hemolytic potential was unaltered upon amidation. The selectivity index revealed that these peptides are more selective to bacteria than mammalian cells. Cytotoxicity against Hep3B cells reveals their potential to destroy cancer cells; they showed potential inhibition compared to anticancer drug silymarin. The study also highlights the need for further truncations and modifications of esculentin peptides for developing them as lead drug molecules.

Genetic structure and demographic history of Indirana semipalmata, an endemic frog species of the Western Ghats, India.

The evolutionary potential of a species mainly depends on the level of genetic variation in their populations. Maintenance of gene variation enables populations to adapt more quickly to environmental changes. The geographical gaps also influence the distribution and evolutionary history of many mountain frogs in the world. Hence, a sound knowledge in population genetic structure of a species will help understand its population dynamics and develop conservation strategies. In the context of facing threats to the amphibian fauna of Western Ghats due to habitat loss, we used both mitochondrial and nuclear DNA markers to investigate the genetic structure of an endemic frog species of the Western Ghats (Indirana semipalmata) with restricted distribution. The present study showed the importance of mountain gaps in shaping the species' structuring in the Western Ghats. Though a high genetic diversity was observed for the species when considering a single unit in the southern Western Ghats, the restricted gene flow on/between either side of the Shencottah gap with genetic clustering of the sampled populations may warrant a unique management plan for the species. The habitat fragmentation of the Western Ghats through anthropogenic activities may result in severe setbacks to the survival of the species in the future.

Decoding the proteomic changes involved in the biofilm formation of Enterococcus faecalis SK460 to elucidate potential biofilm determinants.

BACKGROUND: Enterococcus faecalis is a major clinically relevant nosocomial bacterial pathogen frequently isolated from polymicrobial infections. The biofilm forming ability of E. faecalis attributes a key role in its virulence and drug resistance. Biofilm cells are phenotypically and metabolically different from their planktonic counterparts and many aspects involved in E. faecalis biofilm formation are yet to be elucidated. The strain E. faecalis SK460 used in the present study is esp (Enterococcal surface protein) and fsr (two-component signal transduction system) negative non-gelatinase producing strong biofilm former isolated from a chronic diabetic foot ulcer patient. We executed a label-free quantitative proteomic approach to elucidate the differential protein expression pattern at planktonic and biofilm stages of SK460 to come up with potential determinants associated with Enterococcal biofilm formation. RESULTS: The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of proteomic data revealed that biofilm cells expressed higher levels of proteins which are associated with glycolysis, amino acid biosynthesis, biosynthesis of secondary metabolites, microbial metabolism in diverse environments and stress response factors. Besides these basic survival pathways, LuxS-mediated quorum sensing, arginine metabolism, rhamnose biosynthesis, pheromone and adhesion associated proteins were found to be upregulated during the biofilm transit from planktonic stages. The selected subsets were validated by quantitative real-time PCR. In silico functional interaction analysis revealed that the genes involved in upregulated pathways pose a close molecular interaction thereby coordinating the regulatory network to thrive as a biofilm community. CONCLUSIONS: The present study describes the first report of the quantitative proteome analysis of an esp and fsr negative non gelatinase producing E. faecalis. Proteome analysis evidenced enhanced expression of glycolytic pathways, stress response factors, LuxS quorum signaling system, rhamnopolysaccharide synthesis and pheromone associated proteins in biofilm phenotype. We also pointed out the relevance of LuxS quorum sensing and pheromone associated proteins in the biofilm development of E. faecalis which lacks the Fsr quorum signaling system. These validated biofilm determinants can act as potential inhibiting targets in Enterococcal infections.

Development and characterization of ten polymorphic microsatellites of the Western Ghats frog Indirana semipalmata and cross amplification in related species.

The genus Indirana Laurent, 1986 is composed of 14 species which are endemic to the Western Ghats of India. We isolated and evaluated ten polymorphic microsatellite markers in 32 individuals of Indirana semipalmata. The cross amplification test was successful in two Indirana (I. brachytarsus and I. yadera) and one Walkerana (W. leptodactyla, previously known as I. leptodactyla) species endemic to the Western Ghats. The identified markers will be useful for further studies on the conservation genetics of endemic frog species of Western Ghats.

Smallest lectin-like peptide identified from the skin secretion of an endemic frog, Hydrophylax bahuvistara.

Lectins are sugar-binding proteins and considered as attractive candidates for drug delivery and targeting. Here, we report the identification of the smallest lectin-like peptide (odorranalectin HYba) from the skin secretion of Hydrophylax bahuvistara which is being the shortest lectin-like peptide identified so far from the frog skin secretion, with 15 amino acid residues. The peptide is the first report from an Indian frog and lacks antimicrobial activity but strongly agglutinate intact human erythrocytes. The sequences at the L-fucose recognizing region is conserved as in other lectins reported from frog skin secretion and could be exploited for specificity and drug targeting properties.

Metataxonomic approach to decipher the polymicrobial burden in diabetic foot ulcer and its biofilm mode of infection.

Chronic diabetic foot is a global burden affecting millions of people, and the chronicity of an ulcer is directly linked to the diverse bacterial burden and its biofilm mode of infection. The bacterial diversity of 100 chronic diabetic ulcer samples was profiled via traditional culturing method as well as metagenomic approach by sequencing the 16S rRNA V3 hyper-variable region on Illumina Miseq Platform (Illumina, Inc., San Diego, CA). All the relevant clinical metadata, including duration of diabetes, grade of ulcer, presence of neuropathy, and glycaemic level, were noted and correlated with the microbiota. The occurrence and establishment of bacterial biofilm over chronic wound tissues was revealed by Fluorescent in situ Hybridization and Scanning Electron Microscopy. The biofilm-forming ability of predominant bacterial isolates was studied via crystal violet assay and Confocal Laser Scanning Microscopy. The dominant phyla obtained from bacterial diversity analysis were Firmicutes, Proteobacteria, and Actinobacteria. The dominant aerobic pathogens identified by culture method are Pseudomonas, Proteus, Enterococcus, and Staphylococcus, whereas high-throughput sequencing revealed heightened levels of Streptococcus and Corynebacterium along with 22 different obligate anaerobes. The biofilm occurrence in chronic diabetic ulcer infection is well analysed. Herein, we illustrate the comprehensive pattern of bacterial infection and identify the community composition of chronic wound pathogenic biofilm.

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.

An Amphibian Host Defense Peptide Is Virucidal for Human H1 Hemagglutinin-Bearing Influenza Viruses.

Although vaccines confer protection against influenza A viruses, antiviral treatment becomes the first line of defense during pandemics because there is insufficient time to produce vaccines. Current antiviral drugs are susceptible to drug resistance, and developing new antivirals is essential. We studied host defense peptides from the skin of the South Indian frog and demonstrated that one of these, which we named "urumin," is virucidal for H1 hemagglutinin-bearing human influenza A viruses. This peptide specifically targeted the conserved stalk region of H1 hemagglutinin and was effective against drug-resistant H1 influenza viruses. Using electron microscopy, we showed that this peptide physically destroyed influenza virions. It also protected naive mice from lethal influenza infection. Urumin represents a unique class of anti-influenza virucide that specifically targets the hemagglutinin stalk region, similar to targeting of antibodies induced by universal influenza vaccines. Urumin therefore has the potential to contribute to first-line anti-viral treatments during influenza outbreaks.

Post-translationally modified frog skin-derived antimicrobial peptides are effective against Aeromonas sobria.

Antimicrobial peptides (brevinin1 HYba1 and brevinin1 HYba2) identified from the skin secretion of an endemic frog species of Western Ghats were studied against fish pathogens. Post-translational modifications such as c-terminal amidation and cyclization of the peptides were enhanced on the activity against Aeromonas sobria. Based on the Minimum inhibitory concentration (3 µM), cyclic amidated brevinin Hyba2 was identified as the most promising antimicrobial agent against A. sobria and can be developed further as a lead drug molecule.

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.

Genetic diversity of an endangered bush frog Pseudophilautus wynaadensis (Jerdon, 1854 "1853") from the south of Palghat gap, Western Ghats, India.

BACKGROUND AND AIMS: Samples of endemic endangered frog species, Pseudophilautus wynaadensis, were assessed with the aid of mitochondrial DNA markers in order to study the pattern of intra specific genetic variation of samples lying south of Palghat gap of the Western Ghats, India. MATERIALS AND METHODS: Partial mitochondrial CO1 and 16S gene sequences were obtained for 21 specimens. RESULTS: Phylogenetic analysis revealed that samples from either side of Palghat gap belonged to a single species. The population of frogs south of Palghat gap showed high haplotype diversity and low nucleotide diversity. The relative gene heterogeneity statistics were low and the gene flow estimates were very high. The AMOVA results showed that 96.05% of the total variations were within the populations. CONCLUSION: It was concluded that high genetic diversity with relatively little geographic differentiation was found in the populations of P. wynaadensis lying south of Palghat gap.

Changes in Inflammatory Markers in Bacterial- and Nifedipine-Induced Gingival Inflammation.

BACKGROUND: Inflammatory gingival enlargement is a common observation in routine dental practice. Drugs like nifedipine, used for hypertension and Porphyromonas gingivalis infection-induced gingival enlargement is of a major concern. The current study aims to identify the inflammatory response of the gingival enlargement by measuring the major inflammatory markers fibrinogen and C-reactive protein (CRP). MATERIALS AND METHODS: Thirty-two patients with nifedipine-induced gingival enlargement, twelve with P. gingivalis infection, and seven with both drug and bacterial infection formed the test group and 16 age- and sex-matched healthy subjects formed the control. Gingival status was assessed, and the gingival index is noted. Fibrinogen and CRP were measured in venous blood. Results of the test and controls were compared using t-test and Pearson correlation. RESULTS: It was found that the changes in the inflammatory markers were statically significant in both fibrinogen levels and the CRP. The inflammation was more pronounced in subjects exposed to the drug and bacterial infection. The gingival index was found directly correlating with both the fibrinogen and the CRP levels. CONCLUSION: The quantitative estimation of both fibrinogen and CRP are good markers for the quantification of inflammation and hence evaluating the risk of coronary heart disease (CHD). Substituting the drug and reducing the bacterial load will help in improving the outcome of the treatment and thereby reducing the risk od CHD.

New distribution records for the critically endangered frog Indirana gundia (Dubois, 1986) from Kerala part of Western Ghats, India.

BACKGROUND: Indirana gundia is one of the critically endangered frog species of Western Ghats, India, and known only from the type locality (Gundya in Karnataka State, India) at an elevation of 200 m Mean Sea Level. We provide data on the geographical distribution of this species using molecular tools. NEW INFORMATION: Our results expand the geographical distribution range of this species about 111 km south up to the northern part of Kerala State and recorded at an elevation ranging from 115 m to 200 m asl.