Denise Cavalcante Hissa

Novel surfactant proteins are involved in the structure and stability of foam nests from the frog Leptodactylus vastus.

SUMMARY Many amphibians lay their eggs in foam nests, which allow the eggs to be deposited out of the water. Analysis of some of these foam nests has revealed that they are a rich source of proteins with unusual primary structures and remarkable surfactant activity, named ranaspumins. The aim of this work was to study the foam nests of the frog Leptodactylus vastus in order to obtain information regarding their composition and function and to improve the understanding of ranaspumins, which are probably a novel class of surfactant proteins. Analyses of the foam fluid composition showed proteins and carbohydrates that presumably are responsible for providing nutrients for the developing tadpoles. Investigation of the function of foam fluid in chemical defence revealed no significant biological activity that could be associated with recognized defence compounds. However, foam fluid presented UV absorbance, suggesting a role in protection against sun damage, which is considered to be one of the possible causes of recently reported amphibian population declines. The foam nests do not prevent the colonization of microorganisms, such as the observed bacterial community of predominantly Gram-positive bacilli. L. vastus foam fluid shows a strong surfactant activity that was associated with their proteins and this activity seems to be due mainly to a protein named Lv-ranaspumin. This protein was isolated by ion-exchange chromatography and found to be a 20 kDa monomeric molecule with the following N-terminal sequence: FLEGFLVPKVVPGPTAALLKKALDD. This protein did not show any match to known proteins or structures, which suggests that it belongs to a new class of surfactant protein.

Unique Crystal Structure of a Novel Surfactant Protein from the Foam Nest of the Frog Leptodactylus vastus.

Breeding by releasing eggs into stable biofoams (“foam nests”) is a peculiar reproduction mode within anurans, fish, and tunicates; not much is known regarding the biochemistry or molecular mechanisms involved. Lv‐ranaspumin (Lv‐RSN‐1) is the predominant protein from the foam nest of the frog Leptodactylus vastus. This protein shows natural surfactant activity, which is assumed to be crucial for stabilizing foam nests. We elucidated the amino acid sequence of Lv‐RSN‐1 by de novo sequencing with mass‐spectrometry and determined the high‐resolution X‐ray structure of the protein. It has a unique fold mainly composed of a bundle of 11 α‐helices and two small antiparallel β‐strands. Lv‐RSN‐1 has a surface rich in hydrophilic residues and a lipophilic cavity in the region of the antiparallel β‐sheet. It possesses intrinsic surface‐active properties, reducing the surface tension of water from 73 to 61 mN m−1 (15 μg mL−1). Lv‐RSN‐1 belongs to a new class of surfactants proteins for which little has been reported regarding structure or function.

Microbial epibionts of the colonial ascidians Didemnum galacteum and Cystodytes sp.

Symbiosis with microorganisms has been well documented for many marine invertebrate taxa. However, knowledge of the diversity of microorganisms associated with ascidians is still limited. This study assessed the microbial epibionts of Didemnum galacteum and Cystodytes sp., two ascidian species collected from the western coast of Ceará state (Brazil), at Dois Coqueiros beach and the port of Pecém, respectively. The microbiota were examined using optical microscopy, followed by subsequent analysis of fingerprinting profiles obtained by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries. The microscopy analysis showed for both ascidians a community comprising cyanobacteria, mainly Prochloron-like species, and diatoms. The DGGE results indicated that D. galacteum hosts a more diverse microbiota when compared to Cystodytes sp. The same analysis also suggested that the diversity of the seawater microbiota was higher than the diversity of the ascidian-associated microbiota. The analysis of the 16S rRNA clone libraries revealed the dominance of Proteobacteria symbionts associated with both ascidians, with Alphaproteobacteria as the major component in D. galacteum and Gammaproteobacteria the major component in Cystodytes sp. The analysis of the clone libraries also revealed the presence of other taxa such as Bacteroidetes, Planctomycetes, Actinobacteria, Cyanobacteria, and uncultured bacteria in D. galacteum, but not in Cystodytes sp. Among the bacteria found to be exclusively associated with the ascidians, none were shared by the two studied hosts. The combined results point to a diverse microbiota associated with the external surface of the ascidians, with a mixed composition including organisms typically found in the surrounding seawater, but also a more specific set of taxa.

Interaction of antimicrobial peptide Plantaricin149a and four analogs with lipid bilayers and bacterial membranes

The amidated analog of Plantaricin149, an antimicrobial peptide from Lactobacillus plantarum NRIC 149, directly interacts with negatively charged liposomes and bacterial membranes, leading to their lysis. In this study, four Pln149-analogs were synthesized with different hydrophobic groups at their N-terminus with the goal of evaluating the effect of the modifications at this region in the peptide’s antimicrobial properties. The interaction of these peptides with membrane models, surface activity, their hemolytic effect on red blood cells, and antibacterial activity against microorganisms were evaluated. The analogs presented similar action of Plantaricin149a; three of them with no hemolytic effect (< 5%) until 0.5 mM, in addition to the induction of a helical element when binding to negative liposomes. The N-terminus difference between the analogs and Plantaricin149a retained the antibacterial effect on S. aureus and P. aeruginosa for all peptides (MIC50 of 19 μM and 155 μM to Plantaricin149a, respectively) but resulted in a different mechanism of action against the microorganisms, that was bactericidal for Plantaricin149a and bacteriostatic for the analogs. This difference was confirmed by a reduction in leakage action for the analogs. The lytic activity of Plantaricin149a is suggested to be a result of the peptide-lipid interactions from the amphipathic helix and the hydrophobic residues at the N-terminus of the antimicrobial peptide.

Crystallization and preliminary X-ray diffraction of the surfactant protein Lv-ranaspumin from the frog Leptodactylus vastus

Lv-ranaspumin is a natural surfactant protein with a molecular mass of 23.5 kDa which was isolated from the foam nest of the frog Leptodactylus vastus. Only a partial amino-acid sequence is available for this protein and it shows it to be distinct from any protein sequence reported to date. The protein was purified from the natural source by ion-exchange and size-exclusion chromatography and was crystallized by sitting-drop vapour diffusion using the PEG/Ion screen at 293 K. A complete data set was collected to 3.5 Å resolution. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.96, b = 89.99, c = 106.00 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54%.

Frog Foam Nest Protein Diversity and Synthesis.

Some amphibian species have developed a breeding strategy in which they deposit their eggs in stable foam nests to protect their eggs and larvae. The frog foam nests are rich in proteins (ranaspumin), especially surfactant proteins, involved in the production of the foam nest. Despite the ecological importance of the foam nests for evolution and species conservation, the biochemical composition, the long-term stability and even the origin of the components are still not completely understood. Recently we showed that Lv-RSN-1, a 23.5-kDa surfactant protein isolated from the nest of the frog Leptodacylus vastus, presents a structural conformation distinct from any protein structures yet reported. So, in the current study we aimed to reveal the protein composition of the foam nest of L. vastus and further characterize the Lv-RSN-1. Proteomic analysis showed the foam nest contains more than 100 of proteins, and that Lv-RSN-1 comprises 45% of the total proteins, suggesting a key role in the nest construction and stability. We demonstrated by Western blotting that Lv-RSN-1 is mainly produced only by the female in the pars convoluta dilata, which highlights the importance of the female preservation for conservation of species that depend on the production of foam nests in the early stages of development. Overall, our results showed the foam nest of L. vastus is composed of a great diversity of proteins and that besides Lv-RSN-1, the main protein in the foam, other proteins must have a coadjuvant role in building and stability of the nest.

First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin-trypsin inhibitor purified from Lonchocarpus sericeus seeds: Effect of Lonchocarpus sericeus protease inhibitor on Aedes aegypti

BACKGROUND Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin-trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases. RESULTS LsCTI showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mass determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (Ki ) for chymotrypsin (8.24 x 10-8 m). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50-percent enzyme inhibition (IC50 ) of LsCTI was 4.7 x 10-7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL-1 , but caused a high larval mortality rate (77%) and delayed development (37%). CONCLUSIONS LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development.