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Multiple oxysterlin peptide sequence alignment. The signal peptide was found up to the A23 position. Oxysterlins 1 and 3 have greater similarity. All cecropins have a high identity of the signal peptide and the amine terminal oxysterlin domain of the mature peptide. Oxysterlin 2 had a middle K36-Q54 region that may be the result of alternative splicing. https://doi.org/10.1371/journal.pone.0187914.g001
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Dung beetles are exposed to a complex microbiological ecosystem during their life cycle. Characterization of novel host-defense peptides (HDP) is essential to understanding the host innate immune response in insects. It constitutes a promising alternative to look for new therapeutic agents against pathogenic microbes. We identified four new HDP, Ox...
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Citations
... The Defensin TcPaSK, artificial derivative of T. castaneum Defensin 3 with higher cationicity and decreased hydrophobicity, can counteract S. aureus growth through membrane permeation, but there is evidence of a possible block of cell division [86]. Oxysterlin 1, 2 and 3 from Oxysternon conspicillatum are active against MDR bacteria, such as Klebsiella pneumoniae and Pseudomonas aeruginosa, but generally not against Gram-positive bacteria with limited hemolytic activity [87]. Furthermore, an isoform of Cecropin B called CecB Q53 showed promising ability to contain P. aeruginosa, E. coli and Staphylococcus, with high stability to pH, salinity and temperature variations, showing no significant cytotoxic or hemolytic activity [88]. ...
Several insects are known as vectors of a wide range of animal and human pathogens
causing various diseases. However, they are also a source of different substances, such as the
Antimicrobial Peptides (AMPs), which can be employed in the development of natural bioactive
compounds for medical, veterinary and agricultural applications. It is well known that AMP activity,
in contrast to most classical antibiotics, does not lead to the development of natural bacterial resistance,
or at least the frequency of resistance is considered to be low. Therefore, there is a strong interest
in assessing the efficacy of the various peptides known to date, identifying new compounds and
evaluating possible solutions in order to increase their production. Moreover, implementing AMP
modulation in insect rearing could preserve insect health in large-scale production. This review
describes the current knowledge on insect AMPs, presenting the validated ones for the different
insect orders. A brief description of their mechanism of action is reported with focus on proposed
applications. The possible effects of insect diet on AMP translation and synthesis have been discussed.
... Cecropins, α-helical AMPs, are indiscriminately active against Gram negative bacteria, such as E. coli, Klebsiella pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa [75][76][77], or Gram positive bacteria, such as Staphylococcus and Bacillus species [75][76][77]. Their expression can be induced by both Gram positive and negative bacteria [78]: for example, in Lepidoptera, different microbial infections result in different patterns of cecropin gene expression, indicating that various signaling pathways can contribute to the same immune gene expression. ...
... Cecropins, α-helical AMPs, are indiscriminately active against Gram negative bacteria, such as E. coli, Klebsiella pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa [75][76][77], or Gram positive bacteria, such as Staphylococcus and Bacillus species [75][76][77]. Their expression can be induced by both Gram positive and negative bacteria [78]: for example, in Lepidoptera, different microbial infections result in different patterns of cecropin gene expression, indicating that various signaling pathways can contribute to the same immune gene expression. ...
Antimicrobial peptides (AMPs) are a chemically and structurally heterogeneous family of molecules produced by a large variety of living organisms, whose expression is predominant in the sites most exposed to microbial invasion. One of the richest natural sources of AMPs is insects which, over the course of their very long evolutionary history, have adapted to numerous and different habitats by developing a powerful innate immune system that has allowed them to survive but also to assert themselves in the new environment. Recently, due to the increase in antibiotic-resistant bacterial strains, interest in AMPs has risen. In this work, we detected AMPs in the hemolymph of Hermetia illucens (Diptera, Stratiomyidae) larvae, following infection with Escherichia coli (Gram negative) or Micrococcus flavus (Gram positive) and from uninfected larvae. Peptide component, isolated via organic solvent precipitation, was analyzed by microbiological techniques. Subsequent mass spectrometry analysis allowed us to specifically identify peptides expressed in basal condition and peptides differentially expressed after bacterial challenge. We identified 33 AMPs in all the analyzed samples, of which 13 are specifically stimulated by Gram negative and/or Gram positive bacterial challenge. AMPs mostly expressed after bacterial challenge could be responsible for a more specific activity.
... They were important participants in most living species' defensive systems, directly killing pathogens and boosting the immune response (Deptuła et al. 2018;Pantic et al. 2017). So far, approximately 3000 natural AMPs have been found in a variety of taxa, notably plants (Retzl et al. 2020), insects (Johanna Toro Segovia et al. 2017), fungi (Nakamura et al. 2017), fishes (Chen et al. 2020), frogs (Barran et al. 2020), and mammals (Mardirossian et al. 2018). AMPs are reportedly less susceptible to drug resistance than conventional antibiotics due to their unique structures and mechanisms of action (Almaaytah et al. 2017;Wei et al. 2018). ...
A recombinant version of the AGAAN antimicrobial peptide (rAGAAN) was cloned, expressed, and purified in this study. Its antibacterial potency and stability in harsh environments were thoroughly investigated. A 15 kDa soluble rAGAAN was effectively expressed in E. coli. The purified rAGAAN exhibited a broad antibacterial spectrum and was efficacious against seven Gram-positive and Gram-negative bacteria. The minimal inhibitory concentration (MIC) of rAGAAN against the growth of M. luteus (TISTR 745) was as low as 60 µg/ml. Membrane permeation assay reveals that the integrity of the bacterial envelope is compromised. In addition, rAGAAN was resistant to temperature shock and maintained a high degree of stability throughout a reasonably extensive pH range. The bactericidal activity of rAGAAN ranged from 36.26 to 79.22% in the presence of pepsin and Bacillus proteases. Lower bile salt concentrations had no significant effect on the function of the peptide, whereas higher concentrations induced E. coli resistance. Additionally, rAGAAN exhibited minimal hemolytic activity against red blood cells. This study indicated that rAGAAN may be produced on a large scale in E. coli and that it had an excellent antibacterial activity and sufficient stability. This first work to express biologically active rAGAAN in E. coli yielded 8.01 mg/ml at 16 °C/150 rpm for 18 h in Luria Bertani (LB) medium supplemented with 1% glucose and induced with 0.5 mM IPTG. It also assesses the interfering factors that influence the activity of the peptide, demonstrating its potential for research and therapy of multidrug-resistant bacterial infections.
... Cecropins are one of the largest groups of insects AMPs and HDPs. Nevertheless, a few functional Coleoptera cecropins have been identified in species like Acalolepta luxuriosa (Cec) [30]; Oxysternon conspicillatum (oxysterlins) [25], Paederus dermatitis (Sarcotoxin Pd) [31] and, Tribolium castaneum, where only non-functional cecropins (pseudogenes) had been reported [22]. ...
... Therefore, to live in these environments a highly efficient immune system that probably relies on the expression of diverse and potent HDPs and AMPs is necessary. Other Coleoptera and Scarabaeidae AMPs, like Acaloleptin, Sarcotoxin, Coprisin and Oxysterlins, have been characterized with antimicrobial activities [23][24][25]. To investigate further the diversity of HDPs and AMPs expressed by Scarabaeidae, we have previously used a transcriptome-based strategy to identify new HDPs expressed by Dichotomius satanas and Onthophagus curvicornis (Coleoptera: Scarabaeidae) identifying three new putative antimicrobial peptides named Satanin 1, 2 and Curvicin [32]. ...
... Only a few cecropins are currently described in dung beetles (Coleoptera: Scarabaeidae), with a highly conserved N-terminal domain and considerably differ from the Diptera and Lepidoptera cecropins [26]. The Oxysterlins [25] are the most similar peptides at the amino acid sequence level with our peptides. Oxysterlin 3 has an identity percentage of 82 %, 89 %, and 79 % with Satanin 1, 2, and Curvicin, respectively. ...
Antibiotic resistance is an increasing global problem and therapeutic alternatives to traditional antibiotics are needed. Antimicrobial and host defense peptides represent an attractive source for new therapeutic strategies, given their wide range of activities including antimicrobial, antitumoral and immunomodulatory. Insects produce several families of these peptides, including cecropins. Herein, we characterized the sequence, structure, and biological activity of three cecropins called satanin 1, 2, and curvicin, found in the transcriptome of two dung beetle species Dichotomius satanas and Onthophagus curvicornis. Sequence and circular dichroism analyses show that they have typical features of the cecropin family: short length (38 - 39 amino acids), positive charge, and amphipathic α-helical structure. They are active mainly against Gram-negative bacteria (3.12 - 12.5 μg/mL), with low toxicity on eukaryotic cells resulting in high therapeutic indexes (TI > 30). Peptides also showed effects on TNFα production in LPS-stimulated PBMCs. The biological activity of Satanin 1, 2 and Curvicin makes them interesting leads for antimicrobial strategies.
... So far, the identification of insect HDP has focused on Hemiptera, Hymenoptera, Lepidoptera, and Diptera orders (Mylonakis et al. 2016). In Coleoptera, the most diverse order of insects, only a few HDP have been reported (Cociancich et al. 1994, Bulet et al. 2004, Mylonakis et al. 2016, Toro Segovia et al. 2017. Insect HDP have been classified in different families in accordance with their sequence, physico-chemical and structural properties. ...
... The HDP reported in the Scarabaeidae family are scarce compared with their wide diversity of species, consisting of over 30,000 globally. The sequence and function of only a few of these peptides have been characterized, including some defensin and cecropin families, like Coprisin and Oxysterlin (Tomie et al. 2003, Lee et al. 2014, Toro Segovia et al. 2017. Therefore, this work sought to identify and describe new putative HDP in publicly available assembled transcript sequences from the NCBI Transcriptome Shotgun Assembly (TSA) database of seven different species of Scarabaeidae and two new transcriptomes from the neotropical beetles Dichotomius satanas and Ontophagus curvicornis, both species widely distributed inhabiting the Andean region of Colombia (Bouchard et al. 2011, Cultid-Medina et al. 2012). ...
... Queries lists from different HDP InterPro families were constructed (Cecropin: IPR020400; Defensin: IPR017982; Coleoptericin: IPR009382, and Attacin: IPR005520 IPR005521; Supp Table S1 [online only]); the Cecropin family was complemented with Oxysterlins (Toro Segovia et al. 2017). With the TSA BLAST databases and the HDP queries, a multi-TBLASTn search was constructed and the resulting sequences were filtered according to the E-score ≤ 0.01 (Altschul et al. 1990). ...
The Coleoptera Scarabaeidae family is one of the most diverse groups of insects on the planet, which live in complex microbiological environments. Their immune systems have evolved diverse families of Host Defense Peptides (HDP) with strong antimicrobial and immunomodulatory activities. However, there are several peptide sequences that await discovery in this group of organisms. This would pave the way to identify molecules with promising therapeutic potential. This work retrieved two sources of information: 1) De-novo transcriptomic data from two species of neotropical Scarabaeidae (Dichotomius satanas and Ontophagus curvicornis); 2) Sequence data deposited in available databases. A Blast-based search was conducted against the transcriptomes with a subset of sequences representative of the HDP. This work reports 155 novel HDP sequences identified in nine transcriptomes from seven species of Coleoptera: D. satanas (n = 76; 49.03%), O. curvicornis (n = 23; 14.83%), (Trypoxylus dichotomus) (n = 18; 11.61%), (Onthophagus nigriventris) (n = 10; 6.45%), (Heterochelus sp) (n = 6; 3.87%), (Oxysternon conspicillatum) (n = 18; 11.61%), and (Popillia japonica) (n = 4; 2.58%). These sequences were identified based on similarity to known HDP insect families. New members of defensins (n = 58; 37.42%), cecropins (n = 18; 11.61%), attancins (n = 41; 26.45%), and coleoptericins (n = 38; 24.52%) were described based on their physicochemical and structural characteristics, as well as their sequence relationship to other insect HDPs. Therefore, the Scarabaeidae family is a complex and rich group of insects with a great diversity of antimicrobial peptides with potential antimicrobial activity.
... Oxysterlins 1, 2, and 3, isolated from the dung beetles O. conspicillatum (Toro Segovia et al., 2017), similar to Cecropin A, Aedesin, Lucilin and HKABF, were found selective for Gm -ve bacteria and efficiently kill multidrug resistant (MDR) strains, including E. coli ESBL, E. cloacae, S. typhi, and E. coli with MIC values between 3.12 and 50 µg/mL. Cecropins were first isolated from the silk moth H. cecropia. ...
... et al., 2016;Prajapati et al., 2020). Briefly, in silico analysis, able to find out the vital function with their encoded genetic factors from the large sequence (Toro Segovia et al., 2017). On the other hand, the modification of natural peptides in laboratory conditions also provides massive success in achieving the desired goal of improving activity and pharmacokinetics profiles. ...
Biofilms form a complex layer with defined structures, that attach on biotic or abiotic surfaces, are tough to eradicate and tend to cause some resistance against most antibiotics. Several studies confirmed that biofilm-producing bacteria exhibit higher resistance compared to the planktonic form of the same species. Antibiotic resistance factors are well understood in planktonic bacteria which is not so in case of biofilm producing forms. This may be due to the lack of available drugs with known resistance mechanisms for biofilms. Existing antibiotics cannot eradicate most biofilms, especially of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). Insects produce complex and diverse set of chemicals for survival and defense. Antimicrobial peptides (AMPs), produced by most insects, generally have a broad spectrum of activity and the potential to bypass the resistance mechanisms of classical antibiotics. Besides, AMPs may well act synergistically with classical antibiotics for a double-pronged attack on infections. Thus, AMPs could be promising alternatives to overcome medically important biofilms, decrease the possibility of acquired resistance and treatment of multidrug-resistant pathogens including ESKAPE. The present review focuses on insect-derived AMPs with special reference to anti-biofilm-based strategies. It covers the AMP composition, pathways and mechanisms of action, the formation of biofilms, impact of biofilms on human diseases, current strategies as well as therapeutic options to combat biofilm with antimicrobial peptides from insects. In addition, the review also illustrates the importance of bioinformatics tools and molecular docking studies to boost the importance of select bioactive peptides those can be developed as drugs, as well as suggestions for further basic and clinical research.
... In the case of O. conspicillatum, they push dung portions instead of forming a ball. As larvae, O. conspicillatum feed on organic material present in the dung portion such as fungi and decaying organic matter (Toro Segovia et al. 2017). Although one of the most common species in baited pitfall traps, many details of their life cycle are still unknown, with existing biological and ecological details based entirely on field observations (Edmonds and Zidek 2004) because laboratory rearing so far has proven difficult. ...
... They observed that isolated brood cells in the laboratory were highly contaminated by pathogens, a situation that did not occur in the presence of adults or in the field. This is consistent with findings of antimicrobial substances in the fat body of O. conspicillatum (Toro Segovia et al. 2017) which have multiple functions such as a resource to synthetize proteins and metabolites (Arrese and Soulages 2010). It is still unknown whether parental care lasts until the new adults emerge. ...
Aspects of life history and host interactions of Megalolaelaps colossus were studied in a laboratory environment. These mites appear to require a host for survival, but hosts do not survive very long in a laboratory setting, leading to a modified rearing protocol relying on regular host replacement. Specific data on phenology and incidence are reported. Direct observations on feeding, mating, transmission, and interaction with other mites could not be obtained, but indirect observations allow some hypotheses in each of these areas.
... In the domesticated silkworm Bombyx mori, the Cec gene family is composed of at least 14 elements (two Cec A (A1 and A2), six Cec B (B1-B6), one Cec C, two Cec D (D and D2), one Cec E, and two enbocins (enb 1 and 2)), organized in two clusters, mapping on two different chromosomes [72]. In Coleoptera, functional Cec genes have been identified in species like Acalolepta luxuriosa (Cec; [20]), Oxysternon conspicillatum (Oxysterlins; [19]), and Paederus dermatitis (Sarcotoxin Pd; [21]), whereas only non-functional Cec pseudogenes have been reported in the coleopteran model Tribolium castaneum [73,74]. ...
... Several natural Cecs and Cec-analogs have also shown activity against the food-borne Gram-negative pathogen Salmonella typhimurium, included in the high priority group of the WHO list (e.g., [19,23,25,58,80,98]). In addition, some dipteran Cec AMPs, such as those from the mosquitos Aedes albopictus and Culex pipens, were active against Francisella novicida, a facultative Gram-negative bacterium used as reference species to model F. tularensis, a zoonotic pathogen causing tularemia in humans and animals [28]. ...
... (including K. pneumoniae and E. coli), bacterial species belonging to the WHO first critical group. M. domestica Mdc, black fly SibaCec, and dung beetle Oxysterlins were active against MDR and clinically isolated E. coli strains[19,33,108], while H. cecropia Cec A and P. xuthus Papiliocin efficiently killed MDR P. aeruginosa isolates ...
The alarming escalation of infectious diseases resistant to conventional antibiotics requires urgent global actions, including the development of new therapeutics. Antimicrobial peptides (AMPs) represent potential alternatives in the treatment of multi-drug resistant (MDR) infections. Here, we focus on Cecropins (Cecs), a group of naturally occurring AMPs in insects, and on synthetic Cec-analogs. We describe their action mechanisms and antimicrobial activity against MDR bacteria and other pathogens. We report several data suggesting that Cec and Cec-analog peptides are promising antibacterial therapeutic candidates, including their low toxicity against mammalian cells, and anti-inflammatory activity. We highlight limitations linked to the use of peptides as therapeutics and discuss methods overcoming these constraints, particularly regarding the introduction of nanotechnologies. New formulations based on natural Cecs would allow the development of drugs active against Gram-negative bacteria, and those based on Cec-analogs would give rise to therapeutics effective against both Gram-positive and Gram-negative pathogens. Cecs and Cec-analogs might be also employed to coat biomaterials for medical devices as an approach to prevent biomaterial-associated infections. The cost of large-scale production is discussed in comparison with the economic and social burden resulting from the progressive diffusion of MDR infectious diseases.
... A kinetic time-kill experiment was performed based on a modified method of Segovia et al. [19,20]. Two standard S. aureus strains were separately grown in MH broth medium with a final count of approximately 1 × 10 5 CFU/mL. ...
Background:
Methicillin-resistant Staphylococcus aureus (MRSA) causes serious infections in hospitals. Penthorum chinense Pursh (PCP), employed by the Miao ethnic minority in China, presents antibacterial activities. In this study, the anti-Staphylococcus aureus activities in the pinocembrin-7-O residue-rich fraction from PCP (PGF) were evaluated and characterized.
Methods:
The PGF was prepared with 70% ethanol reflux extraction followed by fractional extraction and column chromatography. Pinocembrin-7-O residue components were identified with electrospray ionization mass spectrometry (ESI-MS). Anti-S. aureus activities of the fraction and the main components were evaluated in vitro with serially diluted microbroth assays. Cytotoxicity was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) chromogenic assays using the NCTC 1469 cell line.
Results:
This study indicated that the PGF and three components (S1, S2, and S3) presented anti-S. aureus activities, including against clinically isolated MRSA strains. The molecular masses of S1, S2, and S3 were identical to those of pinocembrin-7-O-[4″,6″-hexahydroxydiphenoyl (HHDP)]-β-D-glucose, pinocembrin-7-O-[3″-O-galloyl-4″,6″-(s)-HHDP]-β-D-glucose, and Thonningianin A, respectively. The PGF, S1, S2, and S3 all presented an identical minimum inhibitory concentration (MIC) against S. aureus ATCC 25923 and ATCC 43300, which was 62.5 μg/mL. The minimum bactericidal concentrations (MBCs) of the PGF and S3 against ATCC 25923 were 125 and 250 μg/mL, and the MBCs of the PGF, S2, and S3 against ATCC 43300 were 250, 500, and 250 μg/mL, respectively. A time-kill assay consistently indicated that none of the bacterial clones of ATCC 25923 and ATCC 43300 could survive under 2× and 4× MIC PGF treatment for 24 h, respectively. In contrast, 104 CFU (colony-forming units) of ATCC 25923 and ATCC 43300 were killed by 8× and 4× MIC S3 within 24 h, respectively. Additionally, 1×, 2×, and 4× MIC the PGF presented similar postantibiotic effects (PAEs) on the strain ATCC 25923. However, the PAE of the PGF on the strain ATCC 43300 was concentration dependent (1× < 2× < 4× MIC). Finally, the PGF (200 μg/mL) and S3 (60 μg/mL) showed no cytotoxicity against human hepatoma cells.
Conclusions:
The PGF and S3 from PCP present potential for the treatment of S. aureus and MRSA infections. The components S1 and S2 present inhibition activities against S. aureus.
... animals and bacteria. AMPs display broad-spectrum antimicrobial activity; melittin [2], cecropin [3] and magainin [4] have been shown to be active against bacteria. In many investigations, moderate hydrophobicity and net positive charge were essential for the antimicrobial activity of AMPs. ...
Developing novel antimicrobial agents is a top priority in fighting against bacterial resistance. Thus, a series of new monomer and dimer peptides were designed and synthesized by conjugating fatty acids at the N-terminus of partial d-amino acid substitution analogues of anoplin and dimerization. The new peptides exhibited more efficient killing of gram-negative and gram-positive bacteria, including methicillin-resistant Staphylococcus aureus compared with the parent peptide anoplin, and the dimer peptides were superior to the monomer peptides. It was important that the new peptides displayed low impact on bacterial resistance development. In addition, the antimicrobial activities were not significantly influenced by a physiological salt environment. They also presented high stability in the presence of protease or serum. Almost all of the new peptides had better selectivity towards anionic bacterial membranes over zwitterionic mammalian cell membranes. Moreover, the new peptides displayed synergistic or additive effects when used together with the antibiotics rifampicin and polymyxin B. These results showed that the new peptides could also prevent the formation of bacterial biofilms. Furthermore, outer/inner membrane permeabilization and cytoplasmic membrane depolarization experiments revealed that the new peptides had strong membrane permeabilization and depolarization. Confocal laser scanning microscopy, flow cytometry analysis and scanning electron microscopy further demonstrated that the new peptides could damage the integrity of the bacterial membrane. Finally, a DNA-binding affinity assay showed that the new peptides could bind to bacterial DNA. In summary, the conjugation of fatty acids at the N-terminus of peptides and dimerization are promising strategies for obtaining potent antimicrobial agents.
