Antimicrobial action of Phylocaullis boraceiensis mucus

Antimicrobial action of Phylocaullis boraceiensis mucus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antimicrobial action of Phylocaullis boraceiensis mucus Ronaldo Zucatelli Mendonça, renan Araujo, Juliana Aparecida Souza, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7049133/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antimicrobial peptides obtained from invertebrates are good candidates for natural antibiotics due to their broad spectrum of action and low susceptibility to inducing antimicrobial resistance. In the absence of a specific immune system, invertebrates have an efficient innate immune system, composed, among others, of several antimicrobial peptides to defend themselves against pathogenic microorganisms. In this work, we identified and characterized 12 substances in the mucus of P. boraceiensis slugs with potent antimicrobial effects against bacteria and/or yeasts ( P. aeruginosa, E. coli, M. luteus C. albicans ). These substances were isolated by chromatography and mass spectrometry. Two substances obtained (based with on the largest quantity of material) were analyzed in a database, relating them to already known proteins and peptides and their antimicrobial activities. Later, new analyses will be carried out to characterize the other substances discovered in this study. Antimicrobial peptides Slug Bioprospection Phylocaullis boraceiensis Mucus De novo sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Bacterial and fungal infections significantly impact human health, causing high morbidity and mortality. Fungal infections, including those from Candida , Aspergillus , and Cryptococcus , pose serious threats, particularly in immunocompromised individuals (Kathiravan et al., 2012 ). The rise of invasive fungal infections, including those caused by Fusarium and Scedosporium , further complicates treatment (Sable, Strohmaier, & Chodakewitz, 2008 ). Antibiotic resistance, driven by misuse and prolonged therapies, exacerbates bacterial infections (Giedraitiene et al., 2011). Similarly, antifungal treatments face limitations, with resistance emerging even to newer agents like voriconazole and echinocandins (Sable, Strohmaier, & Chodakewitz, 2008 ; Odds et al., 2003 ). Thus, developing new antimicrobials is critical (Kathiravan et al., 2012 ). Natural products remain vital in drug discovery, with many clinical drugs originating from them (Paterson & Anderson, 2005 ; Coelho et al., 2015 ; Toledo-Piza et al., 2016 , 2018 ; Carvalho et al., 2017 , 2022 ; Hayashida et al., 2019 ; Carmona et al., 2020; Mendonça et al., 2024 ). Antimicrobial peptides (AMPs), found in invertebrates, vertebrates, and even bacteria like Lactobacillus , offer broad-spectrum activity against resistant pathogens (Fusetani, 2010 ; Tincu & Taylor, 2004 ; da Silva et al., 2023 ; Sathyan et al., 2012 ). Gastropods, living in contaminated environments, produce mucus containing AMPs like achacin and mitimacin, which exhibit antimicrobial properties (Iijima, Kisugi, & Yamazaki, 2003; Zhong et al., 2013; Hayashida & da Silva Jr, 2021a, 2021b). Our study focuses on Phyllocaulis boraceiensis , a shell-less slug whose mucus has shown antiviral effects (Toledo-Piza et al., 2018 ). This work aims to identify and characterize AMPs in P. boraceiensis mucus with antimicrobial and antifungal potential. 2. Material and Methods 2.1. Animals Specimens of the land slug Phyllocaulis boraceiensis from captive breeding at the Malacology Laboratory of the Butantan Institute were used to collect mucus. The procedures for creating and maintaining the specimens used were carried out according to Toledo-Piza ( 2007 and 2012 ). The animals were kept in plastic nurseries containing fresh soil, with a screened lid, in a pollution-free laboratory environment, with temperature control at ± 24°C and 85% relative air humidity. The animals were fed with different vegetables as recommended by Santos and Thomé ( 2000 ) and the ponds were cleaned every two days. 2.2. Obtaining the crude extract of P. boraceiensis mucus Each mucus sample was stimulated through mechanical processes and obtained from four specimens, which were free on a smooth surface (Petri dish) containing a thin layer of 0.06% saline solution (NaCl) for approximately 5 minutes. This technique aims to facilitate the release and collection of mucus. The samples were stored in a freezer at – 80°C. (Toledo-Piza, 2012 ). 2.3 Extraction solubilization and chromatography The total mucus suspended in collection solution (0.06% NaCl) was diluted 1:1 in 0.1% trifluoroacetic acid (TFA). The extraction of molecules retained in the solid phase was done by sonication in eight sections of 15 seconds each, interspersed for 60 seconds resting at a frequency of approximately 10 Hz in an ice bath. The sample was then kept in an ice bath for 30 min. on the magnetic stirrer and centrifuged for 30 min. at 16,000 x g, 4°C.Then, the supernatant was collected and applied to a Sep-Pak C 18 cartridge equilibrated with 0.05% trifluoroacetic acid (TFA). An elution was performed using 80% acetonitrile in 0.05% TFA. The fraction corresponding to the eluted phase was lyophilized resuspended in acidified ultrapure water (TFA 0.05%), and subjected to high performance liquid chromatography (HPLC) using the UFLC Shimadzu system with column of semi-preparative reverse phase type JUPITER C18 with coupled UV reader (250 mm .5 mL/min. The manually collected fractions were freeze-dried and reconstituted in Mili-Q water. 2.4 Determination of antimicrobial activity of fractions To verify the antimicrobial activity of the fractions, the growth inhibition assay in liquid media was used (Bulet et al., 1993 ). The microorganisms used to test the fractions obtained by chromatography were Gram-negative bacteria of the species Escherichia coli SBS363 and Pseudomonas aeruginosa ATCC27853, Gram-positive bacteria of the species Candida albicans MDM8. The strains were obtained from the Special Applied Toxinology Laboratory of the Butantan Institute, where they are kept in a freezer at -80 °C and periodically harvested. The assays were carried out in 96-well plates with an aliquot of 20 µl of each fraction and 80 µl of microorganisms in their culture medium, at a final concentration of 10 4 cells/ml of yeast or 10 5 cells/ml of bacteria in each well. The microorganisms of the C. albicans MDM8 strain were resuspended in culture medium containing potato dextrose at a concentration of 12 g/L. The bacteria E. coli, P. aeruginosa and M. luteus were resuspended in a nutrient-poor medium (PB: peptone 10g/L; NaCl 5g/L; pH 7.4). Wells containing only medium, medium and microorganisms and medium and antibiotic (penicillin/streptomycin 10mg/ml). They were used respectively as a control for contamination of the medium, and as negative and positive controls for the growth of microorganisms. The absorbance measurement (λ = 595 nm) of the culture was carried out on a Victor 3 microplate reader after 18 hours of incubation at 30 °C, in order to evaluate microbial growth through the turbidity of the environment. The fractions that inhibited the growth of the tested microorganisms were screened based on the apparent expressiveness of inhibition (reduction in turbidity) and subjected to mass spectrometry to verify homogeneity. 2.5 Polyacrylamide gel electrophoresis Proteins were analyzed by polyacrylamide gel electrophoresis in the presence or absence of SDS (Laemmli, 1970 ), using 10 cm x 10 cm x 1.5 mm mini gels in a Digel DGV-10 tank. Samples were diluted in the same volume of sample buffer (4 times concentrated) (350 mM Tris/HCl, 30% glycerol; 1.2 mg bromophenol blue). The material was applied to a 4.5% stacking gel and 6% polyacrylamide separation gel and subjected to electrophoresis with a constant current of 80 volts. The gels were stained with Coomassie blue R-250. The molecular weight marker Tools was used. 2.6 Reduction, alkylation and trypsinization 2.6.1. Digestion “in solution” The reduction and alkylation of Cys residues present in the molecules makes it possible to evaluate the presence of disulfide bonds in peptides purified from mucus. To this, 10 µL aliquot of the dry fraction was dissolved in 20 µL of 0.4 M NH 4 HCO 2 . 5 µL of 45nM dithiothreitol (DTT) was added to this solution. The sample was kept at 50 °C for 15 minutes. After being cooled to room temperature, 5 µL of 100 nM iodoketamine was added and kept at room temperature for 15 min, protected from light. 130 µL of Milli-Q water were added to dilute the Urea and 2 µL of trypsin, incubated at 37 °C for 12 hours. To stop the reaction, 160 µL of 0.1% acidified water (TFA) was added. The product was concentrated in a vacuum centrifuge and desalination in Zip Tip C18 columns. 2.6.2. “In gel” digestion The bands present in the SDS-PAGE gels were cut and subjected to the in-gel digestion process according to the protocol described by (Hanna et al., 2000 ) with some modifications. Initially, the cut gel fragments were incubated in 500 µL of a 50% methanol solution in ultrapure water containing 5% acetic acid for 2h. This solution was then removed by aspiration with a pipette and 500 µL was added to the gel fragments, which remained in this solution for another hour. The fragments were then dehydrated by incubating for 10 min (twice for 5 min) in 200 µL of acetonitrile (100%). The acetonitrile was aspirated with a pipette and the remainder was evaporated in a vacuum concentration system (speed vac). After this step, the gel fragments were rehydrated for 30 min in 30 µL (per gel fragment) of the reducing solution (10 mM dithiothreitol in 100 mM ammonium bicarbonate). Then the alkylating solution was removed by aspiration with a pipette and the gel fragments were subjected to incubation for 10 min in 100 µL (per gel fragment) of 100 mM ammonium bicarbonate. After this step, the ammonium bicarbonate solution was removed and 200 µL (per gel fragment) of acetonitrile (100%) were added and the gel fragments remained incubated in this solution for 5 min. The acetonitrile was removed, and the gel fragments were again incubated for 10 min in 200 µL of 100 M ammonium bicarbonate solution. In the last stage of dehydration, the ammonium bicarbonate solution was removed and the gel fragments were incubated for 10 min (twice for 5 min) in 200 µL of 100% acetonitrile The acetonitrile solution was aspirated with a pipette and the remainder was evaporated in a speed vac system. The gel fragments were rehydrated in 16 µL of a fresh trypsin solution (50 mg/ µL in 50 mM ammonium bicarbonate), in an ice bath for 30 min. Then the trypsin solution was removed, an ammonium bicarbonate solution was added (in sufficient volume to cover the gel fragments) and the tubes containing the gel fragments were incubated in an oven at 37 °C for 18 hours. The peptides were extracted from the gel by adding 30 µL (per gel fragment) of solution 1 ( 5% formic acid in ultrapure water) and incubating the fragments for 10 min at room temperature. After this, the solution containing the peptides was transferred to a new tube and added 12 µL (per gel fragment) of solution 2 (5% formic acid in 50% acetonitrile). The gel fragments were incubated for 20 min (twice for 10 min) in this solution, which was subsequently transferred to the tube containing the peptides extracted with solution 1. The solution was concentrated in speed vac and the resulting peptides were resuspended in 15 µL (per sample) of 0.1% formic acid. 2.7 Mass spectrometry The peptides were resuspended in 20 µL of ACN/H 2 O 1:1 with formic acid and subjected to analysis by direct infusion in a Thermo Scientific LTQ-Orbitrap LC/MS under a flow rate of 0.5 µL/min in a 50 µL syringe. The fragmentation of mono or doubly charged ions was carried out under different collision energies (Ecol) against inert gas (argon) so that the best MS/MS spectra could be used in sequencing. DIC spectra against inert gas (Ar) molecules were acquired every 0.5 second and the final spectra composition was obtained after 1.5 min of acquisition. 2.8 Bioinformatics analysis The m/Z (mass/charge ratio) values ​​corresponding to the y and b series were recorded using the Peaks application. The data (files with “.raw” extension) referring to the MS/MS analyzes using the LTQ spectrometer were loaded into the Peaks program, through which deconvolution of the m/z values ​​was performed to obtain the molecular mass as well as sequencing analysis of probable peptides present in the sample. The fragments that contain the N-terminal portion of the molecule are from the “b” series, while those that contain the C-terminal part of the molecule are from the “y” series, so the difference in mass between adjacent fragments of the same series corresponds to the mass of the amino acid residue present in that position of the molecule. The sequenced peptides were subjected to searches for similarities with other proteins in public databases using the BLAST program (2013) ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) with the Protein blast tool, PSI algorithm -BLAST and taxonomic filter for the phylum Mollusca and in APD2 peptide databases (Wang and Wang, 2004). 3. Results 3.1 Purification and antimicrobial effect of fractions This study was carried out with the mucus of five specimens of slugs of the species P. boraceiensis . The molecules present in this pool were separated on a C-18 Sep-Pak column. The fraction eluted at 80% ACN was subjected to HPLC (high performance liquid chromatography) using a semi-preparative column. The eluted fractions ( Fig. 1 ) were collected manually and tested for the growth inhibition effect of different microbial strains: E. coli, P. aeruginosa and C. albicans. The fractions that showed an antimicrobial effect against any of the tested microorganisms were concentrated within an ACN gradient range between 31% and 63%. According to Table 1 , seven fractions were obtained that inhibited the growth of the Gram-negative bacterium P. aeruginosa ATCC27853: Fr48, Fr54, Fr58, Fr65, Fr 67, Fr72 and Fr79; four fractions that inhibited E. coli growth: Fr47, Fr48, Fr58 and Fr60; seven fractions that inhibited M. luteus growth: Fr54, Fr65, Fr72, Fr73, Fr74, Fr79 and Fr84; six fractions that inhibited the growth of C. albicans : Fr48, Fr54, Fr65, Fr67, Fr74, Fr79; Red arrows indicate peaks referring to the fractions selected for characterization ( Fig. 1 ). Table 1 Antimicrobial activity spectrum of P. boraceiensis mucus fractions obtained by high performance liquid chromatography (HPLC). Growth inhibition detected (+); Growth inhibition not detected (-). ACN (%): % of acetonitrile to each fraction on HPLC on linear gradient. Fraction Time (min) ACN (%) P. aeruginosa E. coli M. luteus C. albicans Fr47 44,4 32 - + - - Fr48 44,9 33 + + - + Fr54 47,5 36 + - + + Fr58 48,7 38 + + - - Fr60 50,8 40 - + - - Fr65 53,4 46 + - + + Fr67 55,0 48 + - - + Fr72 58,5 49 + - + - Fr73 59,6 52 - - + - Fr74 60,1 53 - - + + Fr79 63,4 58 + - + + Fr84 65,7 62 - - + - 3.2 Detection of proteins in fractions 67 and 72 For subsequent purification and sequencing, two active fractions were selected based with on the largest quantity of material to be submitted to characterization studies: Fr 67 and Fr 72 ( Fig. 2 ). Polyacrylamide gel electrophoresis was performed with aliquots of the 67 and 72. As can be observed in Fig. 2 , two bands is visible in both fractions, one with 13 kDa (t5, t3), and other weighing approximately 19 kDa (t4,t2), being more intense in fraction 67. 3.2.1 Sequencing of t2, t3, t4 and t5 of bands from fractions 67 and 72. Aliquots of Fr 67 and Fr 72 were digested in solution with trypsin and bands t2, t3, t4 and t5 were digested with trypsin in gel. The digested samples were analyzed on an LTQ spectrometer Spectra generated from the digested samples were deconvoluted and resequenced using the Peaks program (Figs. 3 – 8 ). To measure the reliability of the sequencing of a given amino acid obtained from the mass spectrum and its deconvolution, the Peaks program uses a value (score) expressed as a percentage called Local confidence (LC). In a given sequence, from the sum of the LC values ​​for each sequenced amino acid, the TLC (Total local confidence) value is obtained. By dividing the TLC value by the number of amino acids obtained in the sequence, the ALC (Average local confidence) value is obtained, which is the index used by the software to determine how reliable a given sequence obtained through the sequencing tool. The sequences obtained with the highest ALC in each fraction were adopted as the most probable sequences ( Table 2 ). In the Fr 67 fraction, the most likely fragment obtained was the CCDVCFK sequence. The Fr 72 fraction and bands t2 and t5 presented the FLKQLAR sequence as the most likely fragment. In fraction t4 , the most likely fragment obtained was the MLKQLAR sequence. Fraction t3 presented the sequence MYELANLVNELLR as the most likely fragment. The four sequences considered presented an ALC value greater than 90% ( Table 2 ). Table 2 – Most likely sequences of the fractions 67 and 72 and bands t2, t3, t4 and t5 . The sequences were obtained from the deconvolution of mass spectra using the Peaks software tool. In each sample, the sequence considered most likely was the one with the highest ALC value. *Higher ALC. Sample MS Sequence * ALC (%) Fr 67 CCDVCFK 99 t4 (~ 19 kDa) MLKQLAR 98 t5 (~ 13 kDa) FLKQLAR 96 Fr 72 97 t2 (~ 19 kDa) 91 t3 (~ 13 kDa) MYELANLVNELLR 97 3.4 Sequencing of peptides and proteins found in fractions 67 and 72 The four selected sequences ( Table 2 ) , obtained by sequencing both the t2, t3, t4 and t5 bands digested by trypsin in gel and the fractions 67 and 72, were searched in databases using the BLAST protein tool (BLAST: Basic Local Alignment Search Tool”. The result of non-predictive or hypothetical protein with the highest total score was adopted as the most relevant result ( Table 3 ). Table 3 – Result of the comparison of the sequences found in the fractions 67 and 72 fractions and bands t2, t3, t4 and t5 with the BLAST database. The alignment considered most relevant was the non-predictive/hypothetical protein with the highest total score. * Result of non-predictive/hypothetical sequence with highest total score. Sequênce BLAST* Score Total Origen CCDVCFK Toll-like receptor m 24.8 Mytilus galloprovincialis MLKQLAR Coiled-coil domain-containing protein 17 26.1 Crassostrea gigas FLKQLAR Adenosine deaminase AGSA precursor 21.5 Aplysia californica MYELANLVNELLR Multiple epidermal growth factor-like domains 10 27.8 Crassostrea gigas The CCDVCFK sequence fragment, obtained from Fr 67, showed 100% identity with the fragment located between amino acids 18 and 23 of the toll-like m receptor from Mytilus galloprovincialis (Toubiana et al., 2013). The FLKQLAR sequence fragment, obtained from Fr 72, t2 and t5, showed 100% identity with the fragment located between amino acids 476 and 481 of the precursor protein of the AGSA adenosine deaminase enzyme from Aplysia californica (Akalal et al., 2003 ). The MLKQLAR sequence fragment, obtained from t4, showed 100% identity with the fragment located between amino acids 849 and 855 of the coiled-coil 17 carrier protein from Crassostrea gigas (Zhang et al., 2012 ). The sequence fragment MYELANLVNELLR , obtained from t3, showed 80% identity with the fragment located between amino acids 178 and 187 of a protein carrying domains similar to multiple epidermal growth factors 10, from Crassostrea gigas (Zhang et al., 2012 ) ( Table 4 ). Table 4 – Comparison of the sequences found in the fractions 67 and 72 and t2, t3, t4 3 t5 bands with the APD2 peptide database. *Only the results with the greatest similarity were considered. Sequência APD2 Similaridade (%) Fonte CCDVCFK Patelamida D 30,00 Lissoclinum patella MLKQLAR Mastoparana-VT7 38,46 Vespa tropica FLKQLAR Temporina-Rb 41,66 Rana ridibunda MYELANLVNELLR Temporina-1PRb 42,85 Rana pirica The four sequences selected from Fr 67, Fr 72, t2, t3, t4 and t5 were searched in the APD2 peptide database (Wang, Li and Wang 2009 , Wang and Wang 2004). The CCDVCFK sequence fragment, obtained from Fr 67, showed 30% similarity with the antimicrobial peptide (PAM) Patelamide D, originating from Lissoclinum patella (Degnan et al., 1989). The FLKQLAR sequence fragment, obtained from Fr 72, t2 and t5, showed 41.66% similarity with the PAM Temporin-Rb, from Rana ridibunda (Asoodeh, Zardini and Chamani, 2012). The MLKQLAR sequence fragment, obtained from t4, showed 38.46% similarity with the Mastoparan-VT7 PAM, originating from Vespa tropica (Yang et al., 2013). The sequence fragment MYELANLVNELLR , obtained from t3, showed 42.85% similarity with the PAM Temporin-1PRb, originating from Rana pirica (Conlon et al., 2004). 4. Discussion The present work aimed to elucidate molecules with antimicrobial effects in the mucus of the slug P. boraceiensis. After purifying the mucus by liquid chromatography and antimicrobial assay, growth inhibition was observed by twelve fractions in at least one of the strains used. The two fractions chosen for characterization, Fr 67 and Fr 72, were shown to be capable of inhibiting the growth of P. aeruginosa, C. albicans , and P. aeruginosa and M. luteus r espectively ( Fig. 3 ) After polyacrylamide gel electrophoresis, four proteins were revealed: two in Fr 67 with around 13 and 19 kDa in weight respectively; and two in Fr 72, also weighing about 13 and 19 kDa respectively ( Fig. 4 ). After trypsin digestion and de novo sequencing of the Fr 67 and Fr 72 fractions and their derived bands t2, t3, t4 and t5, the most likely sequenced peptide from each fraction and band was considered (See item 3.3) which resulted in a list of four distinct peptides. Two bands derived from different fractions, t5 of Fr 67 and t2 of Fr 72, most likely presented a peptide with the same sequence: FLKQLAR . Sequencing carried out from the total digested Fr 72 fraction also showed FLKQLAR as the most likely sequence. Thus, it was verified that the sequence found consists of both a fragment of a protein of approximately 13 kDa present in Fr 67 and a fragment of a protein of approximately 19 kDa present in Fr 72. After being submitted to the BLAST database, it showed 100% similarity with a fragment located between amino acids 476 and 481 of the precursor protein of the adenosine deaminase enzyme (AGSA) from gastropods of the Aplysia californica species. Renamed MGDF by Akalal et al., ( 2003 ), AGSA is an abundant protein in A. californica eggs and plays an important role in the development of the central nervous system of embryos. Gastropod eggs of the species Aplysia kurodai were discovered as being a source of the peptide Aplisianin E, which showed antimicrobial activity against fungi of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans (Iijima, Kisugi and Yamazaki 1995). Another antimicrobial peptide (Aplisianin A), was isolated by Kamiya et al., 1986 ) from the albumin gland of gastropods of the same species, showing antimicrobial activity on bacteria of the species Bacillus subtilis. If the proteins in bands t2 and t5 are influential factors in the antimicrobial effects observed in Fr 67 and Fr 72 and the FLKQLAR fragment is related to the observed antimicrobial activities, it would have an influence on both yeasts and Gram-negative and Gram-positive bacteria (Table 1 ). Studies are necessary to elucidate the identity and specific effect of the proteins found. The most likely peptide obtained from Fr 67, whose sequence is CCDVCFK , was not found among the sequences obtained from bands t4 and t5, which suggests that CCDVCFK represents or constitutes a peptide weighing less than 10 kDa. When subjected to a search in the BLAST database, it showed 100% identity with the fragment located between amino acids 18 and 23 of the toll-like receptor m from the mussel Mytilus galloprovincialis. Among metazoans, toll-like receptors, as well as their correlate, Toll, can function both as cytokine receptors and pathogen recognition receptors (PAMPs) (Toubiana et al., 2013). Furthermore, it is known that toll-like receptors are involved in controlling the expression of antimicrobial peptides in invertebrates (Tauszig et al., 2000 ). Thus, it is suggested that the toll-like receptor-like peptide found in the Fr 67 fraction, in addition to being involved in promoting an antimicrobial effect through the control of expression of antimicrobial peptides, may also have a direct antimicrobial effect, if it is an influential molecule in the observed effect. in the Fr 67 fraction. More studies are needed to elucidate the peptide in question, as well as its function. Constituting the protein of around 19 kDa found in the Fr 67 fraction, the most likely peptide MLKQLAR sequenced from band t4, showed 100% identity with the fragment located between amino acids 849 and 855 of the protein carrying coiled-coil domain 17 originating from the oyster Crassostrea gigas (Gueguen et al., 2006 ; Gonzalez et al, 2007 ) Ryan et al., ( 2013 ) studied antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and antagonists called anti-antimicrobial peptides, demonstrating that antagonists can structurally interact with antimicrobial peptides forming functionally inert helical oligomers. Despite the possible similarity of the protein found in the Fr 67 fraction of mucus with an antimicrobial anti-peptide, more studies are needed to elucidate its function and, eventually, what would be the role of an antimicrobial anti-peptide in mucus, which in principle would have an agonist function in regarding the antimicrobial effect and immunobiological role (Loker et al., 2004 ). The most likely peptide sequenced from band t3, sequence MYELANLVNELLR , constitutes a protein of around 13 kDa present in the Fr 72 fraction (Table 2 ). When submitted to the BLAST database, it showed 80% identity with the fragment located between amino acids 178 and 187 of a protein carrying domains similar to multiple epidermal growth factors 10, originating from the oyster Crassostrea gigas. Multiple epidermal growth factor (MEGF)-like domain-bearing proteins are membrane proteins important for cellular development and communication. However, any relationship between MEGF and antimicrobial effects is unknown. It is therefore suggested that the selected sequence does not present similarity with any known protein with antimicrobial effect if another unstudied sequence from the t3 band does not reveal similarity between the protein of approximately 13 kDa observed in Fr 72, with some protein with known antimicrobial effect. Therefore, it would be possible to assume that the antimicrobial effect observed in Fr 72 would not be related to the protein observed in band t3. More studies are needed to support this hypothesis. When subjected to a search in the APD2 peptide database, the selected sequences showed between 30 and 43% similarity with peptides already studied ( Table 4 ). Although three of the four sequences studied appear to be protein fragments, their comparison to peptide databases is justified, since it is known that PAMs can originate from proteins (Riciluca et al., 2012 ). Patelamide D, a peptide that showed 30% similarity with the CCDVCFK sequence (Table 4 ), is produced by the cyanobacterium of the species Prochloron didemni , a symbiote of the marine benthic invertebrate of the Ascidaceae family, Lissoclinum patella . It is known that patellamides have moderate cytotoxicity and activity against multidrug-resistant cancer cell lines (Degnan et al., 1989, Ireland et al., 1982, Wllliams and Jacobs, 1993). Although direct evidence that Patelamide D has an antimicrobial effect is unknown, studies have shown that some AMPs may also present cytotoxicity against cancer cells (Hoskin and Ramamoorthy, 2008 ). The MLKQLAR sequence showed 38.46% similarity with the Mastoparana-VT7 peptide, originating from a wasp of the Vespa tropica species. Mastoparan-VT7 has antimicrobial activity against Gram-positive bacteria of the species Staphylococcus aureus and Enterococcus faecalis ; Gram-negative bacteria of the species Klebsiella pneumoniae , Bacillus pyocyaneus , Pseudomonas aeruginosa and Escherichia coli ; and yeast of the species C. albicans (Yang et al., 2013). The FLKQLAR sequence fragment, obtained from Fr 72, t2 and t5, showed 41.66% similarity with the Temporin-Rb PAM, originating from the frog Rana ridibunda . Temporin-Rb has an antimicrobial effect against bacteria of the species E. coli , S. dysgalactiae , S. agalactiae , S. aureus , S. typhimurium and K. pneumoniae (Asoodeh, Zardini and Chamani, 2012). The MYELANLVNELLR sequence fragment, obtained from t3, showed 42.85% similarity with the Temporin-1PRb PAM, originating from the frog Rana pirica . Temporin-1PRb has an antimicrobial effect against bacteria of the species E. coli , S. aureus and against yeasts of the species C. albicans (Conlon et al., 2004). 5. Conclusion Four newly sequenced peptides were analyzed from two fractions that showed an antimicrobial effect at the tested concentration of P. boraceiensis mucus . None of the four sequences analyzed, CCDVCFK, MLKQLAR, FLKQLAR and CCDVCFK showed similarity to protein fragments with known direct antimicrobial effects. However, three of the four proteins compared interestingly presented some characteristic that is somehow related to the mechanism of action of antimicrobial peptides, whether representing a receptor involved in the production of AMPs, a protein presenting characteristic domains in antimicrobial peptides or a protein embryonic structure present in eggs, a structure known to contain important antimicrobial peptides. However, it was not possible to establish a relationship between such information and the observed antimicrobial effect. Regarding the similarities observed in peptide databases, three of the four sequences compared showed some similarity with PAMs. The observed similarity may be an indication that the observed fragments constitute peptides with antimicrobial effects. More studies are needed to elucidate this hypothesis. Based on the comparisons carried out in the two databases, it is proposed that it is more likely that the molecules related to the observed antimicrobial effects are peptides and not proteins since the analyzed fragments, even those belonging to proteins, are more related to peptides with antimicrobial activity known. Future studies are necessary to isolate, identify and characterize the molecules present in the mucus fractions of P. boraceiensis with antimicrobial effects. However, this work demonstrated the wealth of molecules with antimicrobial effect for yeasts and Gram positive and negative bacteria in the mucus of P. boraceiensis . Furthermore, information was obtained that may be useful during the continuation of the process of isolation and characterization of antimicrobial factors from P. boraceiensis mucus. Declarations ACKNOWLEDGEMENTS We thanks a Butantan Institute and to Dra. Ana Rita de Toledo-Piza, for the donation of material and guidance in its processing. ETHICS APPROVAL AND CONSENT TO PARTICIPATE This research was approved and performed in accordance with the Ethical Principles in Animal Research adopted by the Ethics Committee in the Use of Animals of Butantan Institute ( I-958-12 ). CONFLICT OF INTEREST The authors declare no financial or commercial conflict of interest. FUNDING This work was supported in part by Butantan Institute , by the Research Support Foundation of the State of São Paulo ( FAPESP/CeTICS ) (Grant No. 2013/07467-1), FAPESP : (Grant No. 2012/22555-1, by the Brazilian National Council for Scientific and Technological Development ( CNPq ) (Grant No. 472744/2012-7), by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil ( CAPES ) - Finance Code 001 and by the Biomedical Sciences Institute - São Paulo University ( ICB-USP ) Author Contributions Conceptualization, R.Z.M and P.I.S.J.; Data curation, R.Z.M and P.I.S.J.; Formal analysis, R.Z.M and P.I.S.J.; Funding acquisition, R.Z.M and P.I.S.J.; Investigation, R.L.A. Methodology, P.I.S.J.; Project administration, R.Z.M and P.I.S.J.; Resources, R.Z.M and P.I.S.J.; Supervision, R.Z.M., Validation, R.Z.M and P.I.S.J.; Writing-original draft, R.L.A. Writing-review and editing, J.A.P.S, R.Z.M and P.I.S.J. 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Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7049133","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487123117,"identity":"44ab8950-1323-4b16-8a24-3daec359ccbd","order_by":0,"name":"Ronaldo Zucatelli Mendonça","email":"","orcid":"","institution":"Instituto Butantan","correspondingAuthor":false,"prefix":"","firstName":"Ronaldo","middleName":"Zucatelli","lastName":"Mendonça","suffix":""},{"id":487123118,"identity":"ab2e4941-77bf-46b8-a5b5-e6ea28f032ac","order_by":1,"name":"renan Araujo","email":"","orcid":"","institution":"Instituto Butantan","correspondingAuthor":false,"prefix":"","firstName":"renan","middleName":"","lastName":"Araujo","suffix":""},{"id":487123119,"identity":"5f24cbad-f329-4272-93e1-0bba1273d2f7","order_by":2,"name":"Juliana Aparecida Souza","email":"","orcid":"","institution":"Instituto Butantan","correspondingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"Aparecida","lastName":"Souza","suffix":""},{"id":487123120,"identity":"52e0124b-ff9c-44bf-a4d7-9b78741c7dcf","order_by":3,"name":"P.I. Silva Junior","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACZgYGA4aKAyhibERoOXOAgYeBgbGBOC0gwNhGihZzdvYHxZXz7iTuZz97/MHPHQx2/RIJbI8r8GixbOYxMDy77VliD09eYmPvGYbkmTMS2A3P4NFicJiHwbBx2+HEHoYcwwbeNoZkgzMH2CQb8Gphf2DYOAeohf+NYeNf4rQwGBg2NgC1SOQYNgNtsTM43oBfC9gvDceeGffceGM4W7ZNIkGyvbHdEJ8Wc/7jzwwbau7ItvfnGHx822Zjz8/MfOwhXocBY8EAiS+R2ICIH5xamB8gC9jjVT4KRsEoGAUjEgAAe5tO5jdIsiAAAAAASUVORK5CYII=","orcid":"","institution":"Instituto Butantan","correspondingAuthor":true,"prefix":"","firstName":"P.I.","middleName":"Silva","lastName":"Junior","suffix":""}],"badges":[],"createdAt":"2025-07-04 19:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7049133/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7049133/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87054891,"identity":"c238d5f2-496e-470a-ba44-f66a9075d772","added_by":"auto","created_at":"2025-07-18 15:32:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51510,"visible":true,"origin":"","legend":"\u003cp\u003ePurification of antimicrobial factors from \u003cem\u003eP. boraceiensis mucus by reversed phase HPLC. P. boraceiensis\u003c/em\u003e mucus analyzed by liquid chromatography on a Jupiter C18 semi preparative column with a linear gradient of acetonitrile from 0 to 80% in acidified water for 60 min, at a flow rate of 1.5 mL/min. Marked fractions showed antimicrobial activity respectively for \u003cstrong\u003eE\u003c/strong\u003e: \u003cem\u003eE. coli\u003c/em\u003e; \u003cstrong\u003eC\u003c/strong\u003e: \u003cem\u003eC. albicans\u003c/em\u003e; M: \u003cem\u003eM. luteus\u003c/em\u003e; Q: \u003cem\u003eP. aeruginosa\u003c/em\u003e. Red arrows indicate peaks referring to the fractions selected for characterization. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAbsorbance was monitored at 225 nm, and antibacterial activity was detected by liquid growth inhibition assay\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/c8f268e7d2068aa5a51e0077.png"},{"id":87054892,"identity":"b48fcef2-8234-4ace-902e-992ecca28b13","added_by":"auto","created_at":"2025-07-18 15:32:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30817,"visible":true,"origin":"","legend":"\u003cp\u003ePolyacrylamide-gel electrophoresis (PAGE) of the \u003cstrong\u003eFr 67\u003c/strong\u003e and \u003cstrong\u003eFr 72\u003c/strong\u003e fractions. The Fr 67 and Fr72 fractions were applied to a 4.5% stacking gel and 6% polyacrylamide separation gel, in the presence of 0.1%. sodium dodecyl sulphate (SDS),and subjected to electrophoresis with a constant current of 80 volts. The gels were stained with Coomassie blue.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/a7e0f69f4aee1a9223c68059.png"},{"id":87057098,"identity":"6242ded5-0041-4371-9b34-428afdcfbb17","added_by":"auto","created_at":"2025-07-18 15:56:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54437,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing of the \u003cstrong\u003eCCDVCFK\u003c/strong\u003e peptide from the \u003cstrong\u003eFr67 fraction\u003c/strong\u003e. A: Mass spectrum with fragments assigned to series b (blue) or series y (red). B: Mass spectrum marked with the intervals between fragments to calculate the amino acid sequence.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/2c8bcfe4485bb1bb2f216d19.png"},{"id":87055566,"identity":"22082295-3974-46a2-986d-2d25ca2f065e","added_by":"auto","created_at":"2025-07-18 15:40:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60173,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing of the \u003cstrong\u003eMLKQLAR\u003c/strong\u003e peptide from \u003cstrong\u003eband t4\u003c/strong\u003e. A: Mass spectrum with fragments assigned to series b (blue) or series y (red). B: Mass spectrum marked with the intervals between fragments to calculate the amino acid sequence\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/bf346a120fe4d48800da4e24.png"},{"id":87055852,"identity":"30c3e822-30b9-4f54-9899-24691fe3925a","added_by":"auto","created_at":"2025-07-18 15:48:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56659,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing of the \u003cstrong\u003eFLKQLAR\u003c/strong\u003e peptide from \u003cstrong\u003eband t5\u003c/strong\u003e. A: Mass spectrum with fragments assigned to series b (blue) or series y (red). B: Mass spectrum marked with the intervals between fragments to determine the amino acid sequence\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/96f98432409f6a8dc7834bba.png"},{"id":87055853,"identity":"1da3c6c0-2405-4b26-beba-318b1b854ca9","added_by":"auto","created_at":"2025-07-18 15:48:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":56176,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing of the \u003cstrong\u003eFLKQLAR\u003c/strong\u003epeptide from the \u003cstrong\u003eFr72 \u003c/strong\u003efraction. A: Mass spectrum with fragments assigned to series b (blue) or series y (red). B: Mass spectrum marked with the intervals between fragments to calculate the amino acid sequence.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/54466577845c7b4d643b0eba.png"},{"id":87054897,"identity":"670edec9-e357-4392-9e91-fcb6f2269a7e","added_by":"auto","created_at":"2025-07-18 15:32:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":57039,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing of \u003cstrong\u003eFLKQLAR\u003c/strong\u003epeptide from band \u003cstrong\u003et2.\u003c/strong\u003e A: Mass spectrum with fragments assigned to series b (blue) or series y (red). B: Mass spectrum marked with the intervals between fragments to calculate the amino acid sequence\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/bcedc3c49cf232568b947e0f.png"},{"id":87054905,"identity":"b13a7d48-c5ab-47f0-89a0-82006057d185","added_by":"auto","created_at":"2025-07-18 15:32:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":63951,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing of \u003cstrong\u003eMYELANLVNELLR\u003c/strong\u003e peptide from \u003cstrong\u003eband t3\u003c/strong\u003e. A: Mass spectrum with fragments assigned to series b (blue) or series y (red). B: Mass spectrum marked with the intervals between fragments to calculate the amino acid sequence\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/ac253143155fb017f7417d28.png"},{"id":96244433,"identity":"a23b8ed9-14be-4a33-9ca6-4c66604b2b68","added_by":"auto","created_at":"2025-11-19 07:18:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2176638,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7049133/v1/27aca585-2fb3-4483-a882-617647ac8f10.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimicrobial action of Phylocaullis boraceiensis mucus","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eBacterial and fungal infections significantly impact human health, causing high morbidity and mortality. Fungal infections, including those from \u003cem\u003eCandida\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, and \u003cem\u003eCryptococcus\u003c/em\u003e, pose serious threats, particularly in immunocompromised individuals (Kathiravan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The rise of invasive fungal infections, including those caused by \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eScedosporium\u003c/em\u003e, further complicates treatment (Sable, Strohmaier, \u0026amp; Chodakewitz, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAntibiotic resistance, driven by misuse and prolonged therapies, exacerbates bacterial infections (Giedraitiene et al., 2011). Similarly, antifungal treatments face limitations, with resistance emerging even to newer agents like voriconazole and echinocandins (Sable, Strohmaier, \u0026amp; Chodakewitz, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Odds et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Thus, developing new antimicrobials is critical (Kathiravan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNatural products remain vital in drug discovery, with many clinical drugs originating from them (Paterson \u0026amp; Anderson, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Coelho et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Toledo-Piza et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Carvalho et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hayashida et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Carmona et al., 2020; Mendon\u0026ccedil;a et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Antimicrobial peptides (AMPs), found in invertebrates, vertebrates, and even bacteria like \u003cem\u003eLactobacillus\u003c/em\u003e, offer broad-spectrum activity against resistant pathogens (Fusetani, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tincu \u0026amp; Taylor, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; da Silva et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sathyan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGastropods, living in contaminated environments, produce mucus containing AMPs like achacin and mitimacin, which exhibit antimicrobial properties (Iijima, Kisugi, \u0026amp; Yamazaki, 2003; Zhong et al., 2013; Hayashida \u0026amp; da Silva Jr, 2021a, 2021b). Our study focuses on \u003cem\u003ePhyllocaulis boraceiensis\u003c/em\u003e, a shell-less slug whose mucus has shown antiviral effects (Toledo-Piza et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis work aims to identify and characterize AMPs in \u003cem\u003eP. boraceiensis\u003c/em\u003e mucus with antimicrobial and antifungal potential.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animals\u003c/h2\u003e\u003cp\u003eSpecimens of the land slug \u003cem\u003ePhyllocaulis boraceiensis\u003c/em\u003e from captive breeding at the Malacology Laboratory of the Butantan Institute were used to collect mucus. The procedures for creating and maintaining the specimens used were carried out according to Toledo-Piza (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e and \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The animals were kept in plastic nurseries containing fresh soil, with a screened lid, in a pollution-free laboratory environment, with temperature control at \u0026plusmn; 24\u0026deg;C and 85% relative air humidity. The animals were fed with different vegetables as recommended by Santos and Thom\u0026eacute; (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and the ponds were cleaned every two days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Obtaining the crude extract of \u003cem\u003eP. boraceiensis\u003c/em\u003e mucus\u003c/h2\u003e\u003cp\u003eEach mucus sample was stimulated through mechanical processes and obtained from four specimens, which were free on a smooth surface (Petri dish) containing a thin layer of 0.06% saline solution (NaCl) for approximately 5 minutes. This technique aims to facilitate the release and collection of mucus. The samples were stored in a freezer at \u0026ndash; 80\u0026deg;C. (Toledo-Piza, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Extraction solubilization and chromatography\u003c/h2\u003e\u003cp\u003eThe total mucus suspended in collection solution (0.06% NaCl) was diluted 1:1 in 0.1% trifluoroacetic acid (TFA). The extraction of molecules retained in the solid phase was done by sonication in eight sections of 15 seconds each, interspersed for 60 seconds resting at a frequency of approximately 10 Hz in an ice bath. The sample was then kept in an ice bath for 30 min. on the magnetic stirrer and centrifuged for 30 min. at 16,000 x g, 4\u0026deg;C.Then, the supernatant was collected and applied to a Sep-Pak C 18 cartridge equilibrated with 0.05% trifluoroacetic acid (TFA). An elution was performed using 80% acetonitrile in 0.05% TFA.\u003c/p\u003e\u003cp\u003eThe fraction corresponding to the eluted phase was lyophilized resuspended in acidified ultrapure water (TFA 0.05%), and subjected to high performance liquid chromatography (HPLC) using the UFLC Shimadzu system with column of semi-preparative reverse phase type JUPITER C18 with coupled UV reader (250 mm .5 mL/min. The manually collected fractions were freeze-dried and reconstituted in Mili-Q water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Determination of antimicrobial activity of fractions\u003c/h2\u003e\u003cp\u003eTo verify the antimicrobial activity of the fractions, the growth inhibition assay in liquid media was used (Bulet et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The microorganisms used to test the fractions obtained by chromatography were Gram-negative bacteria of the species \u003cem\u003eEscherichia coli\u003c/em\u003e SBS363 and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC27853, Gram-positive bacteria of the species \u003cem\u003eCandida albicans\u003c/em\u003e MDM8. The strains were obtained from the Special Applied Toxinology Laboratory of the Butantan Institute, where they are kept in a freezer at -80 \u0026deg;C and periodically harvested.\u003c/p\u003e\u003cp\u003eThe assays were carried out in 96-well plates with an aliquot of 20 \u0026micro;l of each fraction and 80 \u0026micro;l of microorganisms in their culture medium, at a final concentration of 10\u003csup\u003e4\u003c/sup\u003e cells/ml of yeast or 10\u003csup\u003e5\u003c/sup\u003e cells/ml of bacteria in each well. The microorganisms of the \u003cem\u003eC. albicans\u003c/em\u003e MDM8 strain were resuspended in culture medium containing potato dextrose at a concentration of 12 g/L. The bacteria \u003cem\u003eE. coli, P. aeruginosa\u003c/em\u003e and \u003cem\u003eM. luteus\u003c/em\u003e were resuspended in a nutrient-poor medium (PB: peptone 10g/L; NaCl 5g/L; pH 7.4). Wells containing only medium, medium and microorganisms and medium and antibiotic (penicillin/streptomycin 10mg/ml). They were used respectively as a control for contamination of the medium, and as negative and positive controls for the growth of microorganisms. The absorbance measurement (λ\u0026thinsp;=\u0026thinsp;595 nm) of the culture was carried out on a Victor 3 microplate reader after 18 hours of incubation at 30 \u0026deg;C, in order to evaluate microbial growth through the turbidity of the environment. The fractions that inhibited the growth of the tested microorganisms were screened based on the apparent expressiveness of inhibition (reduction in turbidity) and subjected to mass spectrometry to verify homogeneity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Polyacrylamide gel electrophoresis\u003c/h2\u003e\u003cp\u003eProteins were analyzed by polyacrylamide gel electrophoresis in the presence or absence of SDS (Laemmli, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1970\u003c/span\u003e), using 10 cm x 10 cm x 1.5 mm mini gels in a Digel DGV-10 tank. Samples were diluted in the same volume of sample buffer (4 times concentrated) (350 mM Tris/HCl, 30% glycerol; 1.2 mg bromophenol blue). The material was applied to a 4.5% stacking gel and 6% polyacrylamide separation gel and subjected to electrophoresis with a constant current of 80 volts. The gels were stained with Coomassie blue R-250. The molecular weight marker Tools was used.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Reduction, alkylation and trypsinization\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1. Digestion \u0026ldquo;in solution\u0026rdquo;\u003c/h2\u003e\u003cp\u003eThe reduction and alkylation of Cys residues present in the molecules makes it possible to evaluate the presence of disulfide bonds in peptides purified from mucus. To this, 10 \u0026micro;L aliquot of the dry fraction was dissolved in 20 \u0026micro;L of 0.4 M NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e2\u003c/sub\u003e. 5 \u0026micro;L of 45nM dithiothreitol (DTT) was added to this solution. The sample was kept at 50 \u0026deg;C for 15 minutes. After being cooled to room temperature, 5 \u0026micro;L of 100 nM iodoketamine was added and kept at room temperature for 15 min, protected from light. 130 \u0026micro;L of Milli-Q water were added to dilute the Urea and 2 \u0026micro;L of trypsin, incubated at 37 \u0026deg;C for 12 hours. To stop the reaction, 160 \u0026micro;L of 0.1% acidified water (TFA) was added. The product was concentrated in a vacuum centrifuge and desalination in Zip Tip C18 columns.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2. \u0026ldquo;In gel\u0026rdquo; digestion\u003c/h2\u003e\u003cp\u003eThe bands present in the SDS-PAGE gels were cut and subjected to the in-gel digestion process according to the protocol described by (Hanna et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) with some modifications. Initially, the cut gel fragments were incubated in 500 \u0026micro;L of a 50% methanol solution in ultrapure water containing 5% acetic acid for 2h. This solution was then removed by aspiration with a pipette and 500 \u0026micro;L was added to the gel fragments, which remained in this solution for another hour. The fragments were then dehydrated by incubating for 10 min (twice for 5 min) in 200 \u0026micro;L of acetonitrile (100%). The acetonitrile was aspirated with a pipette and the remainder was evaporated in a vacuum concentration system (speed vac). After this step, the gel fragments were rehydrated for 30 min in 30 \u0026micro;L (per gel fragment) of the reducing solution (10 mM dithiothreitol in 100 mM ammonium bicarbonate). Then the alkylating solution was removed by aspiration with a pipette and the gel fragments were subjected to incubation for 10 min in 100 \u0026micro;L (per gel fragment) of 100 mM ammonium bicarbonate. After this step, the ammonium bicarbonate solution was removed and 200 \u0026micro;L (per gel fragment) of acetonitrile (100%) were added and the gel fragments remained incubated in this solution for 5 min. The acetonitrile was removed, and the gel fragments were again incubated for 10 min in 200 \u0026micro;L of 100 M ammonium bicarbonate solution. In the last stage of dehydration, the ammonium bicarbonate solution was removed and the gel fragments were incubated for 10 min (twice for 5 min) in 200 \u0026micro;L of 100% acetonitrile\u003c/p\u003e\u003cp\u003eThe acetonitrile solution was aspirated with a pipette and the remainder was evaporated in a speed vac system. The gel fragments were rehydrated in 16 \u0026micro;L of a fresh trypsin solution (50 mg/ \u0026micro;L in 50 mM ammonium bicarbonate), in an ice bath for 30 min. Then the trypsin solution was removed, an ammonium bicarbonate solution was added (in sufficient volume to cover the gel fragments) and the tubes containing the gel fragments were incubated in an oven at 37 \u0026deg;C for 18 hours.\u003c/p\u003e\u003cp\u003eThe peptides were extracted from the gel by adding 30 \u0026micro;L (per gel fragment) of solution 1 ( 5% formic acid in ultrapure water) and incubating the fragments for 10 min at room temperature. After this, the solution containing the peptides was transferred to a new tube and added 12 \u0026micro;L (per gel fragment) of solution 2 (5% formic acid in 50% acetonitrile). The gel fragments were incubated for 20 min (twice for 10 min) in this solution, which was subsequently transferred to the tube containing the peptides extracted with solution 1. The solution was concentrated in speed vac and the resulting peptides were resuspended in 15 \u0026micro;L (per sample) of 0.1% formic acid.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Mass spectrometry\u003c/h2\u003e\u003cp\u003eThe peptides were resuspended in 20 \u0026micro;L of ACN/H\u003csub\u003e2\u003c/sub\u003eO 1:1 with formic acid and subjected to analysis by direct infusion in a Thermo Scientific LTQ-Orbitrap LC/MS under a flow rate of 0.5 \u0026micro;L/min in a 50 \u0026micro;L syringe. The fragmentation of mono or doubly charged ions was carried out under different collision energies (Ecol) against inert gas (argon) so that the best MS/MS spectra could be used in sequencing. DIC spectra against inert gas (Ar) molecules were acquired every 0.5 second and the final spectra composition was obtained after 1.5 min of acquisition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Bioinformatics analysis\u003c/h2\u003e\u003cp\u003eThe m/Z (mass/charge ratio) values ​​corresponding to the y and b series were recorded using the Peaks application. The data (files with \u0026ldquo;.raw\u0026rdquo; extension) referring to the MS/MS analyzes using the LTQ spectrometer were loaded into the Peaks program, through which deconvolution of the m/z values ​​was performed to obtain the molecular mass as well as sequencing analysis of probable peptides present in the sample. The fragments that contain the N-terminal portion of the molecule are from the \u0026ldquo;b\u0026rdquo; series, while those that contain the C-terminal part of the molecule are from the \u0026ldquo;y\u0026rdquo; series, so the difference in mass between adjacent fragments of the same series corresponds to the mass of the amino acid residue present in that position of the molecule.\u003c/p\u003e\u003cp\u003eThe sequenced peptides were subjected to searches for similarities with other proteins in public databases using the BLAST program (2013) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"http://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the Protein blast tool, PSI algorithm -BLAST and taxonomic filter for the phylum Mollusca and in APD2 peptide databases (Wang and Wang, 2004).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Purification and antimicrobial effect of fractions\u003c/h2\u003e\u003cp\u003eThis study was carried out with the mucus of five specimens of slugs of the species \u003cem\u003eP. boraceiensis\u003c/em\u003e. The molecules present in this pool were separated on a C-18 Sep-Pak column. The fraction eluted at 80% ACN was subjected to HPLC (high performance liquid chromatography) using a semi-preparative column. The eluted fractions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e were collected manually and tested for the growth inhibition effect of different microbial strains:\u003c/p\u003e\u003cp\u003e\u003cem\u003eE. coli, P. aeruginosa and C. albicans.\u003c/em\u003e The fractions that showed an antimicrobial effect against any of the tested microorganisms were concentrated within an ACN gradient range between 31% and 63%.\u003c/p\u003e\u003cp\u003eAccording to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, seven fractions were obtained that inhibited the growth of the Gram-negative bacterium \u003cb\u003eP. aeruginosa\u003c/b\u003e ATCC27853: Fr48, Fr54, Fr58, Fr65, Fr 67, Fr72 and Fr79; four fractions that inhibited \u003cb\u003eE. coli\u003c/b\u003e growth: Fr47, Fr48, Fr58 and Fr60; seven fractions that inhibited \u003cb\u003eM. luteus\u003c/b\u003e growth: Fr54, Fr65, Fr72, Fr73, Fr74, Fr79 and Fr84; six fractions that inhibited the growth of \u003cb\u003eC. albicans\u003c/b\u003e: Fr48, Fr54, Fr65, Fr67, Fr74, Fr79; Red arrows indicate peaks referring to the fractions selected for characterization \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntimicrobial activity spectrum of \u003cem\u003eP. boraceiensis\u003c/em\u003e mucus fractions obtained by high performance liquid chromatography (HPLC). Growth inhibition detected (+); Growth inhibition not detected (-). ACN (%): % of acetonitrile to each fraction on HPLC on linear gradient.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003cp\u003e(min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACN (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eM. luteus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr47\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44,4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr48\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44,9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr54\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e47,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr58\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e48,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr60\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e50,8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e53,4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr67\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e55,0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr72\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e58,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr73\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e59,6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr74\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr79\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63,4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFr84\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e65,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Detection of proteins in fractions 67 and 72\u003c/h2\u003e\u003cp\u003eFor subsequent purification and sequencing, two active fractions were selected based with on the largest quantity of material to be submitted to characterization studies: Fr 67 and Fr 72 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Polyacrylamide gel electrophoresis was performed with aliquots of the 67 and 72.\u003c/p\u003e\u003cp\u003eAs can be observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, two bands is visible in both fractions, one with 13 kDa (t5, t3), and other weighing approximately 19 kDa (t4,t2), being more intense in fraction 67.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Sequencing of t2, t3, t4 and t5 of bands from fractions 67 and 72.\u003c/h2\u003e\u003cp\u003eAliquots of Fr 67 and Fr 72 were digested in solution with trypsin and bands t2, t3, t4 and t5 were digested with trypsin in gel. The digested samples were analyzed on an LTQ spectrometer Spectra generated from the digested samples were deconvoluted and resequenced using the Peaks program (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo measure the reliability of the sequencing of a given amino acid obtained from the mass spectrum and its deconvolution, the Peaks program uses a value (score) expressed as a percentage called Local confidence (LC). In a given sequence, from the sum of the LC values ​​for each sequenced amino acid, the TLC (Total local confidence) value is obtained. By dividing the TLC value by the number of amino acids obtained in the sequence, the ALC (Average local confidence) value is obtained, which is the index used by the software to determine how reliable a given sequence obtained through the sequencing tool.\u003c/p\u003e\u003cp\u003eThe sequences obtained with the highest ALC in each fraction were adopted as the most probable sequences \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e In the \u003cb\u003eFr 67\u003c/b\u003e fraction, the most likely fragment obtained was the \u003cb\u003eCCDVCFK\u003c/b\u003e sequence. The \u003cb\u003eFr 72\u003c/b\u003e fraction and bands t2 and t5 presented the \u003cb\u003eFLKQLAR\u003c/b\u003e sequence as the most likely fragment. In \u003cb\u003efraction t4\u003c/b\u003e, the most likely fragment obtained was the \u003cb\u003eMLKQLAR\u003c/b\u003e sequence. Fraction \u003cb\u003et3\u003c/b\u003e presented the sequence \u003cb\u003eMYELANLVNELLR\u003c/b\u003e as the most likely fragment. The four sequences considered presented an ALC value greater than 90% \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026ndash; Most likely sequences of the \u003cb\u003efractions 67 and 72\u003c/b\u003e and \u003cb\u003ebands t2, t3, t4 and t5\u003c/b\u003e. The sequences were obtained from the deconvolution of mass spectra using the Peaks software tool. In each sample, the sequence considered most likely was the one with the highest ALC value. *Higher ALC.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample MS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequence *\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eALC (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFr 67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCCDVCFK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003et4 (~\u0026thinsp;19 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMLKQLAR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003et5 (~\u0026thinsp;13 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eFLKQLAR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFr 72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003et2 (~\u0026thinsp;19 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003et3 (~\u0026thinsp;13 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMYELANLVNELLR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Sequencing of peptides and proteins found in fractions 67 and 72\u003c/h2\u003e\u003cp\u003eThe four selected sequences \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, obtained by sequencing both the \u003cb\u003et2, t3, t4 and t5\u003c/b\u003e bands digested by trypsin in gel and the fractions 67 and 72, were searched in databases using the BLAST protein tool (BLAST: Basic Local Alignment Search Tool\u0026rdquo;. The result of non-predictive or hypothetical protein with the highest total score was adopted as the most relevant result \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026ndash; Result of the comparison of the sequences found in the \u003cb\u003efractions 67\u003c/b\u003e and \u003cb\u003e72\u003c/b\u003e fractions and \u003cb\u003ebands t2, t3, t4 and t5\u003c/b\u003e with the BLAST database. The alignment considered most relevant was the non-predictive/hypothetical protein with the highest total score. * Result of non-predictive/hypothetical sequence with highest total score.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSequ\u0026ecirc;nce\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBLAST*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eScore\u003c/p\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOrigen\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCCDVCFK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eToll-like\u003c/em\u003e receptor m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eMytilus galloprovincialis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMLKQLAR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCoiled-coil domain-containing protein 17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eCrassostrea gigas\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFLKQLAR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdenosine deaminase AGSA precursor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eAplysia californica\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMYELANLVNELLR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMultiple epidermal growth factor-like domains 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eCrassostrea gigas\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe \u003cb\u003eCCDVCFK\u003c/b\u003e sequence fragment, obtained from Fr 67, showed 100% identity with the fragment located between amino acids 18 and 23 of the toll-like m receptor from \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e (Toubiana et al., 2013). The \u003cb\u003eFLKQLAR\u003c/b\u003e sequence fragment, obtained from Fr 72, t2 and t5, showed 100% identity with the fragment located between amino acids 476 and 481 of the precursor protein of the AGSA adenosine deaminase enzyme from \u003cem\u003eAplysia californica\u003c/em\u003e (Akalal et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The \u003cb\u003eMLKQLAR\u003c/b\u003e sequence fragment, obtained from t4, showed 100% identity with the fragment located between amino acids 849 and 855 of the coiled-coil 17 carrier protein from \u003cem\u003eCrassostrea gigas\u003c/em\u003e (Zhang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The sequence fragment \u003cb\u003eMYELANLVNELLR\u003c/b\u003e, obtained from t3, showed 80% identity with the fragment located between amino acids 178 and 187 of a protein carrying domains similar to multiple epidermal growth factors 10, from \u003cem\u003eCrassostrea gigas\u003c/em\u003e (Zhang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026ndash; Comparison of the sequences found in the \u003cb\u003efractions 67 and 72\u003c/b\u003e and \u003cb\u003et2, t3, t4 3 t5\u003c/b\u003e bands with the APD2 peptide database. *Only the results with the greatest similarity were considered.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSequ\u0026ecirc;ncia\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAPD2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSimilaridade (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFonte\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCCDVCFK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePatelamida D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30,00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eLissoclinum\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003epatella\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMLKQLAR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMastoparana-VT7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38,46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eVespa tropica\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFLKQLAR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemporina-Rb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e41,66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eRana ridibunda\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMYELANLVNELLR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemporina-1PRb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42,85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eRana pirica\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe four sequences selected from Fr 67, Fr 72, t2, t3, t4 and t5 were searched in the APD2 peptide database (Wang, Li and Wang \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Wang and Wang 2004).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe CCDVCFK\u003c/b\u003e sequence fragment, obtained from Fr 67, showed 30% similarity with the antimicrobial peptide (PAM) Patelamide D, originating from \u003cem\u003eLissoclinum patella\u003c/em\u003e (Degnan et al., 1989). The \u003cb\u003eFLKQLAR\u003c/b\u003e sequence fragment, obtained from Fr 72, t2 and t5, showed 41.66% similarity with the PAM Temporin-Rb, from \u003cem\u003eRana ridibunda\u003c/em\u003e (Asoodeh, Zardini and Chamani, 2012). The \u003cb\u003eMLKQLAR\u003c/b\u003e sequence fragment, obtained from t4, showed 38.46% similarity with the Mastoparan-VT7 PAM, originating from \u003cem\u003eVespa tropica\u003c/em\u003e (Yang et al., 2013). The sequence fragment \u003cb\u003eMYELANLVNELLR\u003c/b\u003e, obtained from t3, showed 42.85% similarity with the PAM Temporin-1PRb, originating from \u003cem\u003eRana pirica\u003c/em\u003e (Conlon et al., 2004).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe present work aimed to elucidate molecules with antimicrobial effects in the mucus of the slug \u003cem\u003eP. boraceiensis.\u003c/em\u003e After purifying the mucus by liquid chromatography and antimicrobial assay, growth inhibition was observed by twelve fractions in at least one of the strains used. The two fractions chosen for characterization, Fr 67 and Fr 72, were shown to be capable of inhibiting the growth of \u003cem\u003eP. aeruginosa, C. albicans\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eM. luteus r\u003c/em\u003eespectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter polyacrylamide gel electrophoresis, four proteins were revealed: two in Fr 67 with around 13 and 19 kDa in weight respectively; and two in Fr 72, also weighing about 13 and 19 kDa respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter trypsin digestion and de novo sequencing of the Fr 67 and Fr 72 fractions and their derived bands t2, t3, t4 and t5, the most likely sequenced peptide from each fraction and band was considered (See item 3.3) which resulted in a list of four distinct peptides.\u003c/p\u003e\u003cp\u003eTwo bands derived from different fractions, t5 of Fr 67 and t2 of Fr 72, most likely presented a peptide with the same sequence: \u003cb\u003eFLKQLAR\u003c/b\u003e. Sequencing carried out from the total digested Fr 72 fraction also showed \u003cb\u003eFLKQLAR\u003c/b\u003e as the most likely sequence. Thus, it was verified that the sequence found consists of both a fragment of a protein of approximately 13 kDa present in Fr 67 and a fragment of a protein of approximately 19 kDa present in Fr 72. After being submitted to the BLAST database, it showed 100% similarity with a fragment located between amino acids 476 and 481 of the precursor protein of the adenosine deaminase enzyme (AGSA) from gastropods of the \u003cem\u003eAplysia californica\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003eRenamed MGDF by Akalal et al., (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), AGSA is an abundant protein in \u003cem\u003eA. californica\u003c/em\u003e eggs and plays an important role in the development of the central nervous system of embryos. Gastropod eggs of the species \u003cem\u003eAplysia kurodai\u003c/em\u003e were discovered as being a source of the peptide Aplisianin E, which showed antimicrobial activity against fungi of the species \u003cem\u003eSaccharomyces cerevisiae, Schizosaccharomyces pombe\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e (Iijima, Kisugi and Yamazaki 1995). Another antimicrobial peptide (Aplisianin A), was isolated by Kamiya et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) from the albumin gland of gastropods of the same species, showing antimicrobial activity on bacteria of the species Bacillus subtilis. If the proteins in bands t2 and t5 are influential factors in the antimicrobial effects observed in Fr 67 and Fr 72 and the \u003cb\u003eFLKQLAR\u003c/b\u003e fragment is related to the observed antimicrobial activities, it would have an influence on both yeasts and Gram-negative and Gram-positive bacteria (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Studies are necessary to elucidate the identity and specific effect of the proteins found.\u003c/p\u003e\u003cp\u003eThe most likely peptide obtained from Fr 67, whose sequence is \u003cb\u003eCCDVCFK\u003c/b\u003e, was not found among the sequences obtained from bands t4 and t5, which suggests that \u003cb\u003eCCDVCFK\u003c/b\u003e represents or constitutes a peptide weighing less than 10 kDa. When subjected to a search in the BLAST database, it showed 100% identity with the fragment located between amino acids 18 and 23 of the toll-like receptor m from the mussel \u003cem\u003eMytilus galloprovincialis.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAmong metazoans, toll-like receptors, as well as their correlate, Toll, can function both as cytokine receptors and pathogen recognition receptors (PAMPs) (Toubiana et al., 2013). Furthermore, it is known that toll-like receptors are involved in controlling the expression of antimicrobial peptides in invertebrates (Tauszig et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Thus, it is suggested that the toll-like receptor-like peptide found in the Fr 67 fraction, in addition to being involved in promoting an antimicrobial effect through the control of expression of antimicrobial peptides, may also have a direct antimicrobial effect, if it is an influential molecule in the observed effect. in the Fr 67 fraction. More studies are needed to elucidate the peptide in question, as well as its function.\u003c/p\u003e\u003cp\u003eConstituting the protein of around 19 kDa found in the Fr 67 fraction, the most likely peptide \u003cb\u003eMLKQLAR\u003c/b\u003e sequenced from band t4, showed 100% identity with the fragment located between amino acids 849 and 855 of the protein carrying coiled-coil domain 17 originating from the oyster \u003cem\u003eCrassostrea gigas\u003c/em\u003e (Gueguen et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Gonzalez et al, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eRyan et al., (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) studied antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and antagonists called anti-antimicrobial peptides, demonstrating that antagonists can structurally interact with antimicrobial peptides forming functionally inert helical oligomers. Despite the possible similarity of the protein found in the Fr 67 fraction of mucus with an antimicrobial anti-peptide, more studies are needed to elucidate its function and, eventually, what would be the role of an antimicrobial anti-peptide in mucus, which in principle would have an agonist function in regarding the antimicrobial effect and immunobiological role (Loker et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe most likely peptide sequenced from band t3, sequence \u003cb\u003eMYELANLVNELLR\u003c/b\u003e, constitutes a protein of around 13 kDa present in the Fr 72 fraction (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When submitted to the BLAST database, it showed 80% identity with the fragment located between amino acids 178 and 187 of a protein carrying domains similar to multiple epidermal growth factors 10, originating from the oyster \u003cem\u003eCrassostrea gigas.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMultiple epidermal growth factor (MEGF)-like domain-bearing proteins are membrane proteins important for cellular development and communication. However, any relationship between MEGF and antimicrobial effects is unknown. It is therefore suggested that the selected sequence does not present similarity with any known protein with antimicrobial effect if another unstudied sequence from the t3 band does not reveal similarity between the protein of approximately 13 kDa observed in Fr 72, with some protein with known antimicrobial effect. Therefore, it would be possible to assume that the antimicrobial effect observed in Fr 72 would not be related to the protein observed in band t3. More studies are needed to support this hypothesis.\u003c/p\u003e\u003cp\u003eWhen subjected to a search in the APD2 peptide database, the selected sequences showed between 30 and 43% similarity with peptides already studied \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Although three of the four sequences studied appear to be protein fragments, their comparison to peptide databases is justified, since it is known that PAMs can originate from proteins (Riciluca et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePatelamide D, a peptide that showed 30% similarity with the \u003cb\u003eCCDVCFK\u003c/b\u003e sequence (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), is produced by the cyanobacterium of the species \u003cem\u003eProchloron didemni\u003c/em\u003e, a symbiote of the marine benthic invertebrate of the Ascidaceae family, \u003cem\u003eLissoclinum patella\u003c/em\u003e. It is known that patellamides have moderate cytotoxicity and activity against multidrug-resistant cancer cell lines (Degnan et al., 1989, Ireland et al., 1982, Wllliams and Jacobs, 1993).\u003c/p\u003e\u003cp\u003eAlthough direct evidence that Patelamide D has an antimicrobial effect is unknown, studies have shown that some AMPs may also present cytotoxicity against cancer cells (Hoskin and Ramamoorthy, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cb\u003eMLKQLAR\u003c/b\u003e sequence showed 38.46% similarity with the Mastoparana-VT7 peptide, originating from a wasp of the \u003cem\u003eVespa tropica\u003c/em\u003e species. Mastoparan-VT7 has antimicrobial activity against Gram-positive bacteria of the species \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e; Gram-negative bacteria of the species \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eBacillus pyocyaneus\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e; and yeast of the species \u003cem\u003eC. albicans\u003c/em\u003e (Yang et al., 2013).\u003c/p\u003e\u003cp\u003eThe \u003cb\u003eFLKQLAR\u003c/b\u003e sequence fragment, obtained from Fr 72, t2 and t5, showed 41.66% similarity with the Temporin-Rb PAM, originating from the frog \u003cem\u003eRana ridibunda\u003c/em\u003e. Temporin-Rb has an antimicrobial effect against bacteria of the species \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. dysgalactiae\u003c/em\u003e, \u003cem\u003eS. agalactiae\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. typhimurium\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e (Asoodeh, Zardini and Chamani, 2012).\u003c/p\u003e\u003cp\u003eThe \u003cb\u003eMYELANLVNELLR\u003c/b\u003e sequence fragment, obtained from t3, showed 42.85% similarity with the Temporin-1PRb PAM, originating from the frog \u003cem\u003eRana pirica\u003c/em\u003e. Temporin-1PRb has an antimicrobial effect against bacteria of the species \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e and against yeasts of the species \u003cem\u003eC. albicans\u003c/em\u003e (Conlon et al., 2004).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eFour newly sequenced peptides were analyzed from two fractions that showed an antimicrobial effect at the tested concentration of \u003cem\u003eP. boraceiensis mucus\u003c/em\u003e. None of the four sequences analyzed, \u003cb\u003eCCDVCFK, MLKQLAR, FLKQLAR\u003c/b\u003e and \u003cb\u003eCCDVCFK\u003c/b\u003e showed similarity to protein fragments with known direct antimicrobial effects. However, three of the four proteins compared interestingly presented some characteristic that is somehow related to the mechanism of action of antimicrobial peptides, whether representing a receptor involved in the production of AMPs, a protein presenting characteristic domains in antimicrobial peptides or a protein embryonic structure present in eggs, a structure known to contain important antimicrobial peptides. However, it was not possible to establish a relationship between such information and the observed antimicrobial effect.\u003c/p\u003e\u003cp\u003eRegarding the similarities observed in peptide databases, three of the four sequences compared showed some similarity with PAMs. The observed similarity may be an indication that the observed fragments constitute peptides with antimicrobial effects. More studies are needed to elucidate this hypothesis.\u003c/p\u003e\u003cp\u003eBased on the comparisons carried out in the two databases, it is proposed that it is more likely that the molecules related to the observed antimicrobial effects are peptides and not proteins since the analyzed fragments, even those belonging to proteins, are more related to peptides with antimicrobial activity known.\u003c/p\u003e\u003cp\u003eFuture studies are necessary to isolate, identify and characterize the molecules present in the mucus fractions of \u003cem\u003eP. boraceiensis\u003c/em\u003e with antimicrobial effects. However, this work demonstrated the wealth of molecules with antimicrobial effect for yeasts and Gram positive and negative bacteria in the mucus of \u003cem\u003eP. boraceiensis\u003c/em\u003e. Furthermore, information was obtained that may be useful during the continuation of the process of isolation and characterization of antimicrobial factors from \u003cem\u003eP. boraceiensis\u003c/em\u003e mucus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGEMENTS\u003c/p\u003e\n\u003cp\u003eWe thanks a Butantan Institute and \u0026nbsp; to \u0026nbsp;Dra. Ana Rita de Toledo-Piza,\u0026nbsp;for the donation of material and guidance in its processing.\u003c/p\u003e\n\u003cp\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/p\u003e\n\u003cp\u003eThis research was approved and performed in accordance with the Ethical Principles in Animal Research adopted by the Ethics Committee in the Use of Animals of Butantan Institute (\u003cstrong\u003eI-958-12\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCONFLICT OF INTEREST\u003c/p\u003e\n\u003cp\u003eThe authors declare no financial or commercial conflict of interest.\u003c/p\u003e\n\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by \u003cstrong\u003eButantan Institute\u003c/strong\u003e, by the Research Support Foundation of the State of S\u0026atilde;o Paulo (\u003cstrong\u003eFAPESP/CeTICS\u003c/strong\u003e) (Grant No. 2013/07467-1),\u0026nbsp;\u003cstrong\u003eFAPESP\u003c/strong\u003e: \u0026nbsp;(Grant No.\u0026nbsp;2012/22555-1, \u0026nbsp;by the Brazilian National Council for Scientific and Technological Development (\u003cstrong\u003eCNPq\u003c/strong\u003e) (Grant No. 472744/2012-7), by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (\u003cstrong\u003eCAPES\u003c/strong\u003e) - Finance Code 001 \u0026nbsp;and by the Biomedical Sciences Institute - S\u0026atilde;o Paulo University (\u003cstrong\u003eICB-USP\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, R.Z.M and P.I.S.J.; Data curation, R.Z.M and P.I.S.J.; Formal analysis, R.Z.M and P.I.S.J.; Funding acquisition, R.Z.M and P.I.S.J.; Investigation, R.L.A. Methodology, P.I.S.J.; Project administration, R.Z.M and P.I.S.J.; Resources, R.Z.M and P.I.S.J.; Supervision, R.Z.M., Validation, R.Z.M and P.I.S.J.; Writing-original draft, R.L.A. Writing-review and editing, J.A.P.S, R.Z.M and P.I.S.J.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkalal, DG., Bottenstein, JE., Lee, SH., Han, JH., Chang, DJ., Kaang, BK. and Nagle, GT. Aplysia mollusk-derived growth factor is a mitogen with adenosine deaminase activity and is expressed in the developing central nervous system. \u003cstrong\u003eMolecular Brain Research\u003c/strong\u003e, v. 117, n. 2, p. 228\u0026ndash;236, 7 out. \u003cstrong\u003e2003\u003c/strong\u003e. \u003c/li\u003e\n\u003cli\u003eAssodeh, A., Zardini, HZ. and Chamani, J. 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A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica. \u003cstrong\u003ePeptides\u003c/strong\u003e, v. 39, p. 1\u0026ndash;5, jan. \u003cstrong\u003e2013\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial peptides, Slug, Bioprospection, Phylocaullis boraceiensis, Mucus, De novo sequencing","lastPublishedDoi":"10.21203/rs.3.rs-7049133/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7049133/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial peptides obtained from invertebrates are good candidates for natural antibiotics due to their broad spectrum of action and low susceptibility to inducing antimicrobial resistance. In the absence of a specific immune system, invertebrates have an efficient innate immune system, composed, among others, of several antimicrobial peptides to defend themselves against pathogenic microorganisms. In this work, we identified and characterized 12 substances in the mucus of \u003cem\u003eP. boraceiensis\u003c/em\u003e slugs with potent antimicrobial effects against bacteria and/or yeasts (\u003cem\u003eP. aeruginosa, E. coli, M. luteus C. albicans\u003c/em\u003e). These substances were isolated by chromatography and mass spectrometry. Two substances obtained (based with on the largest quantity of material) were analyzed in a database, relating them to already known proteins and peptides and their antimicrobial activities. Later, new analyses will be carried out to characterize the other substances discovered in this study.\u003c/p\u003e","manuscriptTitle":"Antimicrobial action of Phylocaullis boraceiensis mucus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 15:32:31","doi":"10.21203/rs.3.rs-7049133/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3c6b3ef-c569-4096-9fef-cb53be4bba2b","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T14:23:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 15:32:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7049133","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7049133","identity":"rs-7049133","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}