Phyllomedusa Distincta Research Articles

ACT-107, a novel variant of AmpC β-lactamase from Enterobacter huaxiensis isolated from Neotropical leaf frog (Phyllomedusa distincta) inhabiting the Brazilian Atlantic Forest

The aim of this study was to characterise a broad-spectrum cephalosporin-resistant AmpC-positive Enterobacter huaxiensis colonising the skin of a Neotropical frog (Phyllomedusa distincta) inhabiting the Brazilian Atlantic Forest. During a genomic surveillance study of antimicrobial resistance, we screened skin samples from P. distincta. Gram-negative bacteria growing on MacConkey agar plates supplemented with 2 µg/mL ceftriaxone were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. A cephalosporin-resistant E. huaxiensis was sequenced using the Illumina NextSeq platform. Genomic data were analysed using bioinformatics tools, whereas AmpC β-lactamase was characterised in depth by comparative analysis of amino acids, in silico modelling, and analysis of susceptibility to β-lactam antibiotics and combinations of β-lactamase inhibitors. Whole-genome sequencing analysis revealed a novel variant of AmpC β-lactamase belonging to the ACT family, designated ACT-107 by NCBI. This variant contains 12 novel amino acid mutations within the ACT family, 5 in the signal peptide sequence (Ile2, Met14, Tyr16, Gly18 and Thr20), and 7 in the mature protein (Gln22, His43, Cys60, Thr157, Glu225, Ala252 and Asn310). In silico modelling showed that substitutions occurring in the mature chain are localised in the solvent-accessible surface of the protein, where they are not expected to affect the β-lactamase activity, as observed in the resistance profile. Strikingly, 'not designated' ACT variants from E. huaxiensis were clustered (> 96% identity) with ACT-107. Since E. huaxiensis has been isolated from human infection, ACT-107 requires surveillance and the attention of clinicians.

Selection and use of calling site by Boana leptolineata and Phyllomedusa distincta during the reproductive season

ABSTRACT Climatic conditions and microhabitat characteristics, such as the substrate and type of vegetation, influence the choice of male anurans for calling sites that optimize their reproductive success. We evaluated the structure and selection of vocalization microhabitat of 17 individuals of Phyllomedusa distincta Lutz, 1950 and 25 of Boana leptolineata (Braun & Braun, 1977), in a subtropical forest in southern Brazil. We measure the height of the perch, the distance between the perch and the edge of the body of water and the structure of the microhabitat of quadrants used by individuals. The same attributes were measured in the quadrants available (but not used) by the individuals. We classified the microhabitats by visual estimate, assigning percentages of coverage for each quadrant by herbaceous, shrub, tree and wetland vegetation. We observed that both species selected their microhabitat, since the characteristics of the quadrants occupied by the individuals were different from those available. Phyllomedusa distincta was more associated with heterogeneous microhabitats, while B. leptolineata occurred in environments with greater coverage of tree strata. Additionally, we observed that both species used shrubs more frequently as a perch site. The selection of these microhabitat characteristics must be associated with strategies to optimize the use of the reproductive habitat, based on the morphological and behavioral characteristics of the species. Finally, it is possible to infer that the differences observed in the microhabitat structure selected by the species can facilitate the coexistence of both in the context of the heterogeneity of the environment.

The Amphibian Diacylglycerol O-acyltransferase 2 (DGAT2): a ‘paleo-protein’ with Conserved Function but Unique Folding

Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians' skin, and no antibody recognition on fish and mammals' skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a 'paleo-protein'. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding-as indicated by the molecular models and immunohistochemistry analyses-a consequence of the different cysteine pattern but with conserved biological function.

Skin microbiota in frogs from the Brazilian Atlantic Forest: Species, forest type, and potential against pathogens.

The cutaneous microbiota of amphibians can be defined as a biological component of protection, since it can be composed of bacteria that produce antimicrobial compounds. Several factors influence skin microbial structure and it is possible that environmental variations are among one of these factors, perhaps through physical-chemical variations in the skin. This community, therefore, is likely modified in habitats in which some ecophysiological parameters are altered, as in fragmented forests. Our research goal was to compare the skin bacterial community of four anuran species of the Atlantic Forest of Brazil in landscapes from two different environments: continuous forest and fragmented forest. The guiding hypotheses were: 1) microbial communities of anuran skin vary among sympatric frog species of the Atlantic forest; 2) the degree to which forested areas are intact affects the cutaneous bacterial community of amphibians. If the external environment influences the skin microbiota, and if such influences affect microorganisms capable of inhibiting the colonization of pathogens, we expect consequences for the protection of host individuals. We compared bacterial communities based on richness and density of colony forming units; investigated the antimicrobial potential of isolated strains; and did the taxonomic identification of isolated morphotypes. We collected 188 individual frogs belonging to the species Proceratophrys boiei, Dendropsophus minutus, Aplastodiscus leucopygius and Phyllomedusa distincta, and isolated 221 bacterial morphotypes. Our results demonstrate variation in the skin microbiota of sympatric amphibians, but only one frog species exhibited differences in the bacterial communities between populations from fragmented and continuous forest. Therefore, the variation we observed is probably derived from both intrinsic aspects of the host amphibian species and extrinsic aspects of the environment occupied by the host. Finally, we detected antimicrobial activity in 27 morphotypes of bacteria isolated from all four amphibian species.

Assessing Antibacterial Potential of Components of Phyllomedusa distincta Skin and its Associated Dermal Microbiota.

The granular glands of anuran skin secrete an array of bioactive molecules that protect a frog against pathogens and predators. The skin also harbors a microbial community. Although there is evidence to suggest that the microbiota complement the innate immune defense systems against pathogen infection, the effect of the frog bioactive molecules on its resident microbiota has not yet been fully investigated. In the present study, the skin microbiota of Phyllomedusa distincta obtained from two different geographical areas was evaluated with molecular and culture-based approaches. The antagonistic effects exhibited by the host's microbiota and by a novel dermaseptin peptide isolated from P. distincta skin were investigated. Four isolated bacterial colonies displayed antimicrobial activity against known frog pathogens. Our results were consistent with the hypothesis that microbiota from P. distincta may interact with pathogenic microorganisms to protect a frog's health. On the other hand, the novel dermaseptin peptide exhibited an antimicrobial effect on pathogens as well as on some of the bacteria obtained from the skin microbiota. The richness of bacteria on P. distincta skin was further investigated by 16S rRNA gene clone libraries, which revealed that the family Enterobacteriaceae was prevalent, but a high variability at the species level was observed among individual frogs. Differences observed on the microbiota of frogs from contrasting habitats indicated an influence of the environment on the structure of the skin microbiota of P. distincta.

Ancient divergence and recent population expansion in a leaf frog endemic to the southern Brazilian Atlantic forest

The evolutionary history of Neotropical organisms has been often interpreted through broad-scale generalizations. The most accepted model of diversification for the Brazilian Atlantic forest (BAF) rely on putative historical stability of northern areas and massive past habitat replacement of its southern range. Here, we use the leaf frog Phyllomedusa distincta, endemic to the southern BAF, to better understand diversification patterns within this underexplored rainforest region. We used an integrative approach coupling fine-scale sampling and multilocus sequence data, with traditional and statistical phylogeographic (multilocus approximate Bayesian computation) methods to explore alternative hypotheses of diversification. We also employed species paleodistribution modeling to independently verify habitat stability upon a spatially explicit model. Our data support two divergent lineages with coherent geographic distribution that span throughout northern and southern ranges. Demographic estimates suggested the Southern lineage has experienced a recent population expansion, whereas the Northern lineage remained more stable. Hypothesis testing supports a scenario of ancient vicariance with recent population expansion. The paleodistribution model revealed habitat discontinuity during the Last Glacial Maximum (LGM) with one area of putative stability within the range of the Northern lineage. Evidence on genetic structure, demography, and paleodistribution of P. distincta support a historically heterogeneous landscape for the southern BAF, with both areas of forest stability and regions where forest occupation is probably recent. We also associate the southern end of the Cubatao shear zone with a phylogeographic break in the BAF. Taken together, our results argue for the idea of multiple mechanisms generating diversity in this biome and underscore the need of fine-scale data in revealing more detailed pictures.

Cytogenetic analysis of Phyllomedusa distincta Lutz, 1950 (2n = 2x = 26), P. tetraploidea Pombal and Haddad, 1992 (2n = 4x = 52), and their natural triploid hybrids (2n = 3x = 39) (Anura, Hylidae, Phyllomedusinae)

BackgroundNatural polyploidy has played an important role during the speciation and evolution of vertebrates, including anurans, with more than 55 described cases. The species of the Phyllomedusa burmeisteri group are mostly characterized by having 26 chromosomes, but a karyotype with 52 chromosomes was described in P. tetraploidea. This species was found in sintopy with P. distincta in two localities of São Paulo State (Brazil), where triploid animals also occur, as consequence of natural hybridisation. We analyse the chromosomes of P. distincta, P. tetraploidea, and their triploid hybrids, to enlighten the origin of polyploidy and to obtain some evidence on diploidisation of tetraploid karyotype.ResultsPhyllomedusa distincta was 2n = 2x = 26, whereas P. tetraploidea was 2n = 4x = 52, and the hybrid individuals was 2n = 3x = 39. In meiotic phases, bivalents were observed in the diploid males, whereas both bivalents and tetravalents were observed in the tetraploid males. Univalents, bivalents or trivalents; metaphase II cells carrying variable number of chromosomes; and spermatids were detected in the testis preparations of the triploid males, indicating that the triploids were not completely sterile. In natural and experimental conditions, the triploids cross with the parental species, producing abnormal egg clutches and tadpoles with malformations. The embryos and tadpoles exhibited intraindividual karyotype variability and all of the metaphases contained abnormal constitutions. Multiple NORs, detected by Ag-impregnation and FISH with an rDNA probe, were observed on chromosome 1 in the three karyotypic forms; and, additionally, on chromosome 9 in the diploids, mostly on chromosome 8 in the tetraploids, and on both chromosome 8 and 9 in the triploids. Nevertheless, NOR-bearing chromosome 9 was detected in the tetraploids, and chromosome 9 carried active or inactive NORs in the triploids. C-banding, base-specific fluorochrome stainings with CMA3 and DAPI, FISH with a telomeric probe, and BrdU incorporation in DNA showed nearly equivalent patterns in the karyotypes of P. distincta, P. tetraploidea, and the triploid hybrids.ConclusionsAll the used cytogenetic techniques have provided strong evidence that the process of diploidisation, an essential step for stabilising the selective advantages produced by polyploidisation, is under way in distinct quartets of the tetraploid karyotype.

Effects of the substitution of amino acid residues, through chemical synthesis, on the conformation and activity of antimicrobial peptides

Antimicrobial peptides make up an assorted group of molecules which contain from 12 to 50 amino acid residues and which may be produced by microorganisms, plants and animals. From the discovery that these biomolecules are lethal to bacteria, inhibiting the pathogenic organism’s growth, and are also related to innate and adapted defense mechanisms, the investigation of such molecules came to be an emergent research field, in which more than 1800 antimicrobial peptides have so far been discovered throughout the last three decades. These molecules are potential representatives of a new generation of antibiotic agents and the main motivation for such use is their activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria as well as fungi and viruses. An important class of comprising some of these peptides may be found in anurans, from which it has been isolated, a considerable number of antimicrobial peptides with diverse sequences and structures, including linear and dimeric ones. In this work monomeric chains (CH1 e CH2) of the heterodimeric antimicrobial peptide distinctin (isolated in 1999 from Phyllomedusa distincta anurans), as well as its mutated monomers (CH1-S and CH2-S) and the heterodimer itself were synthesized. The distinctin is the peptide with two chains of different sequences (Table 1) bound each other by disulfide bond from the cystein residues constituting the heterodimer. To investigate the effects on the biological activity by amino acids substitution at normal distinctin CH1 and CH2 chains, both were synthesized as well as their similar chains (CH1-S and CH2-S) in which the cystein (Fig.1 a ) residues of each chain were changed by serin residues (Fig. 1 b ). The new chains were named mutants. The synthesis was carried out in solid phase, using Fmoc strategy. The heterodimer distinctin was obtained from CH1 and CH2 chains coupling through cystein residues air oxidation. The results from HPLC purification of the peptides showed different retention times of the normal and mutants chains. In the interaction with biomimetic membranes analyzed by ITC, the CH1, CH2, and distinctin showed unlike behavior. The set of results suggest that the action mechanism of these antimicrobial peptides on the bacterial membrane is intrinsically related with the amino acids sequence and the structural conformation, according to the mechanisms proposed in the literature.

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented 15N and 31P Solid-State NMR Spectroscopy

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with 15N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting 15N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled 31P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.

Análise conformacional por cálculos teóricos da distinctina, peptídeo antimicrobiano isolado de anuros da espécie Phyllomedusa distincta

Various studies demonstrate that different frog species produce distinct classes of biologically active peptides. These peptides can act as alternative agents against pathogenic bacteria and fungi by membrane permeability. Although studies have recently demonstrated that this process is utterly related to the secondary structure adopted by the peptide (in this case, the a-helical structure) when in contact with the bacterial membrane, the detailed mechanism is still unknown. In this work we describe a conformational analysis of distinctin, a heterodimeric peptide isolated from the skin of Phyllomedusa distincta, an anuran found in the Brazilian Atlantic Forest. The study yielded a stable geometry with a high content of the a-helical structure both in chains 1 and 2 of distinctin, showing strong interaction between them.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L −1) than in its absence (93 μmol L −1). Apparent binding constant values were 2160 and 10,000 L mol −1, respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Liposomes fusion and/or lysis investigated by fluorescence and atomic force microscopy

Three dermaseptins, DS 01, DD K, and DD L, were compared with respect to their structural features and interactions with liposomes. Circular dichroic spectra at alcohols of different chain lengths revealed that DS 01 has the higher helicogenic potential in hydrophobic media. Binding of DS 01, DD K, and DD L to liposomes induced significant blue shifts of the emission spectra of the single tryptophan located at position 3 of all sequences indicating association of the peptides with bilayers. Kinetics evaluation of atomic force microscopy images evidenced the strong fusogenic activity of DS 01 whereas DD K and DD L showed increased lytic activities.

A novel heterodimeric antimicrobial peptide from the tree-frog Phyllomedusa distincta

We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree-frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier-transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti-parallel β-sheet. A conformational variation was observed under experimental conditions mimicking a membrane-like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.

Moulting Behavior in Leaf-Frogs of the Genus Phyllomedusa (Anura: Hylidae)

The species belonging to the subfamily Phyllomedusinae presents physiological and behavioral adaptations to economise water such as secretion of lipids coupled with its spreading onto the skin surface through a complex behavior, adoption of diurnal torpor and secretion of uric acid. Here we describe other adaptation probably involved with water economy, registered in Phyllomedusa distincta, P. tarsius, P. tetraploidea and natural hybrids of P. distincta and P. tetraploidea — at dusk, these amphibians perform an elaborate moulting behavior that encompasses the cleaning of the body with the limbs plus gaping and body contractions. Our data show the daily moulting in P. distincta and suggest that the same mechanism occurs for the other species studied. We suppose that daily moulting improves the skin permeability to water being a characteristic of the phyllomedusines which spread lipids onto the skin surface.

Antimicrobial peptides from the Brazilian frog Phyllomedusa distincta1

Different peptides were purified by chromatographic procedures from the skin-secretory glands of the frog Phyllomedusa distincta. These are the first peptides reported from this frog species. Their primary structure was determined by a combination of automated Edman degradation and mass spectrometry. Peptide Q2 contains 25 amino acid residues, peptide Q1 and L have 28 each, peptide M contains 31, and peptide K has 33 amino acid residues. They all showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria, presenting minimal inhibitory concentrations from 0.6 to 40 μM, when tested against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Peptides K, L, and Q1 were chemically synthesized and shown to be active.

Natural Hybridization between Diploid and Tetraploid Species of Leaf-Frogs, Genus Phyllomedusa (Amphibia)

Sympatric populations of the leaf-frogs Phyllomedusa distincta (2n = 26) and P. tetraploidea (4n = 52) were studied in southeastern Brazil. In a region of sympatry, seven of 15 leaf-frogs were triploid hybrids (3n = 39). The advertisement calls of both species are similar, and may not function adequately as a premating isolation mechanism. Triploids apparently exhibit low fertility or sterility, supporting the assertion that diploid and tetraploid populations are valid species. We suggest that P. tetraploidea originated by autopolyploidy of P. distincta, based on the following evidence: (1) the indistinguishable vocalizations of diploid and tetraploid species, and (2) the geographic distribution of species in the Phyllomedusa burmeisteri group.