Ponericins, New Antibacterial and Insecticidal Peptides from the Venom of the Ant Pachycondyla goeldii
The antimicrobial, insecticidal, and hemolytic properties of peptides isolated from the venom of the predatory ant Pachycondyla goeldii, a member of the subfamily Ponerinae, were investigated. Fifteen novel peptides, named ponericins, exhibiting antibacterial and insecticidal properties were purified, and their amino acid sequences were characterized. According to their primary structure similarities, they can be classified into three families: ponericin G, W, and L. Ponericins share high sequence similarities with known peptides: ponericins G with cecropin-like peptides, ponericins W with gaegurins and melittin, and ponericins L with dermaseptins. Ten peptides were synthesized for further analysis. Their antimicrobial activities against Gram-positive and Gram-negative bacteria strains were analyzed together with their insecticidal activities against cricket larvae and their hemolytic activities. Interestingly, within each of the three families, several peptides present differences in their biological activities. The comparison of the structural features of ponericins with those of well-studied peptides suggests that the ponericins may adopt an amphipathic α-helical structure in polar environments, such as cell membranes. In the venom, the estimated peptide concentrations appear to be compatible with an antibacterial activity in vivo. This suggests that in the ant colony, the peptides exhibit a defensive role against microbial pathogens arising from prey introduction and/or ingestion.P82414P82415P82416P82417P82418P82419P82420P82421P82422P82423P82424P82425P82426P82427P82428 The antimicrobial, insecticidal, and hemolytic properties of peptides isolated from the venom of the predatory ant Pachycondyla goeldii, a member of the subfamily Ponerinae, were investigated. Fifteen novel peptides, named ponericins, exhibiting antibacterial and insecticidal properties were purified, and their amino acid sequences were characterized. According to their primary structure similarities, they can be classified into three families: ponericin G, W, and L. Ponericins share high sequence similarities with known peptides: ponericins G with cecropin-like peptides, ponericins W with gaegurins and melittin, and ponericins L with dermaseptins. Ten peptides were synthesized for further analysis. Their antimicrobial activities against Gram-positive and Gram-negative bacteria strains were analyzed together with their insecticidal activities against cricket larvae and their hemolytic activities. Interestingly, within each of the three families, several peptides present differences in their biological activities. The comparison of the structural features of ponericins with those of well-studied peptides suggests that the ponericins may adopt an amphipathic α-helical structure in polar environments, such as cell membranes. In the venom, the estimated peptide concentrations appear to be compatible with an antibacterial activity in vivo. This suggests that in the ant colony, the peptides exhibit a defensive role against microbial pathogens arising from prey introduction and/or ingestion.P82414P82415P82416P82417P82418P82419P82420P82421P82422P82423P82424P82425P82426P82427P82428 high pressure liquid chromatography minimal inhibitory concentration trifluoroethanol Social insects have developed a number of defensive systems that prevent the development of disease within colonies. For example, bee propolis and royal jelly present antimicrobial properties (1Fujiwara S. Imai J. Fujiwara M. Yaeshima T. Kawashima T. Kobayashi K. J. Biol. 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Entomol. 1999; 38: 124-126Crossref Scopus (6) Google Scholar). The mandibular gland secretions of some army ant species also have a dual defensive role against both predators and microbial attacks of brood (8Brown C.A. Watkins J.F. Eldridge D.W. J. Kansas Entomol. Soc. 1979; 52: 119-122Google Scholar). If these mechanisms control the proliferation of many bacteria and fungi in the nesting environment, the introduction of pathogens may also arise from alimentation, especially prey. Among ants, predators stricto sensu are overrepresented within the subfamily Ponerinae (9Hölldobler B. Wilson E.O. The Ants. Springer-Verlag, Berlin1990Crossref Google Scholar). Most of these species capture almost every encountered prey using their venom (10Orivel J. Souchal A. Cerdan P. Dejean A. Sociobiology. 2000; 35: 131-140Google Scholar), which contains peptides (and proteins) (9Hölldobler B. Wilson E.O. The Ants. Springer-Verlag, Berlin1990Crossref Google Scholar, 11Blum M.S. Hermann H.R. Bettini S. Arthropod Venoms. Springer-Verlag, Berlin1978: 801-869Crossref Google Scholar, 12Schmidt J.O. Piek T. Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioural Aspects. Academic Press, London1986: 425-508Crossref Google Scholar). Because these prey are then brought back to the nest immediately after immobilization, their potential infection by bacteria, fungi, or viruses may seriously affect the survival of ant colonies or induce extensive damages because of the high population density combined with the close genetic relationship of the individuals. The antibacterial property of ant venom has only been demonstrated in the fire ant, Solenopsis invicta, in whose venom alkaloids inhibit the growth of both Gram-positive and Gram-negative bacteria and presumably act as a brood antibiotic (12Schmidt J.O. Piek T. Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioural Aspects. Academic Press, London1986: 425-508Crossref Google Scholar, 13Jouvenaz D.P. Blum M.S. MacConnell J.G. Antimicrob. Agents Chemother. 1972; 2: 291-293Crossref PubMed Scopus (72) Google Scholar). Venoms of the wasp,Vespa crabro, honey bees, and various snakes contain antimicrobial peptides, but their functions have not been investigated (14Oren Z. Shai Y. Biochemistry. 1997; 36: 1826-1835Crossref PubMed Scopus (370) Google Scholar, 15Krishnakumari V. Nagaraj R. J. Peptide Res. 1997; 50: 88-93Crossref PubMed Scopus (62) Google Scholar, 16Juvvadi P. Vunnam S. Merrifield R.B. J. Am. Chem. Soc. 1996; 118: 8989-8997Crossref Scopus (104) Google Scholar, 17Blaylock R.S.M. Toxicon. 2000; 38: 1529-1534Crossref PubMed Scopus (26) Google Scholar). Finally, lycotoxins isolated from spider venom are the only antimicrobial peptides in venom for which a preventive role against infections arising from prey ingestion has been demonstrated (18Yan L. Adams M.E. J. Biol. Chem. 1998; 273: 2059-2066Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Here we investigate the possible role of the venom of a predatory ant species in the prevention of microbial disease. The antimicrobial, insecticidal, and hemolytic properties of the venom of the arboreal ponerine ant, Pachycondyla goeldii, were studied. In total, 15 novel peptides, named ponericins, were purified, and their primary structures were fully characterized through amino acid sequencing and matrix-assisted laser ionization/desorption time-of-flight mass spectrometry analyses. According to their amino acid sequences, ponericins were classified into three families named ponericin G, W, and L. Ten peptides were synthesized to perform detailed analyses of their biological activities. The relationships of these peptides with known antimicrobial peptides are discussed. Whole venom reservoirs were dissected from P. goeldiiants collected in Petit Saut, French Guiana. After rinsing in water, they were stored at −20 °C until processed. The venom reservoirs were disrupted by ultrasonic waves in a 30% acetonitrile, 0.2% trifluoroacetic acid solution (10 μl/venom reservoir), and empty reservoirs and membranes were discarded by centrifugation. Whole venom was injected into a C18 reversed-phase column (5 μm, particle size; 220 × 2.1-mm column; Vydac), and separations were performed at a flow rate of 200 μl/min. Peptide elution was monitored at 215 nm. Collected fractions were vacuum-dried and tested for biological activity. First, the venom was separated into four fractions with a gradient consisting of 10–80% solvent B (80% acetonitrile in 0.1% trifluoroacetic acid) for 60 min. Solvent A was 0.1% aqueous trifluoroacetic acid. Second, the venom was further purified on the same column with a biphasic gradient of 10–25% solvent B for 10 min and 25–55% solvent B for 75 min to improve the separation of the peptides contained in the active fraction. The purified peptides were analyzed by matrix-assisted laser ionization/desorption time-of-flight (Voyager Elite; PerSeptive Biosystems, Inc., Framingham, MA) mass spectrometry as described by Seon et al. (19Seon A.A. Pierre T.N. Redeker V. Lacombe C. Delfour A. Nicolas P. Amiche M. J. Biol. Chem. 2000; 275: 5934-5940Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Peptides were sequenced by automated Edman degradation using a Procise pulse-liquid protein sequencer (model 494; PerkinElmer Life Sciences). About 30–60 pmol of peptide were dried and solubilized in 10 μl of 0.1 m ammonium acetate buffer, pH 5.5. Carboxyl-terminal sequences were determined by digestion with carboxypeptidase Y and mass spectrometry analysis of the digestion products as described by Seon et al. (19Seon A.A. Pierre T.N. Redeker V. Lacombe C. Delfour A. Nicolas P. Amiche M. J. Biol. Chem. 2000; 275: 5934-5940Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Ten peptides were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by Synt:em (Nimes, France). Peptides G1, G3, and were to the isolated to some ponericin was synthesized in a with the The peptide was and at the the for of for Because and peptides present the same in their sequence for and for together with differences or but have activity the peptide was further analyzed to an of the biological activity of their in the and hemolytic action of 10 peptides from P. at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested at the tested each the tested concentration was and the to the of the of at the tested in a For each the tested concentration was and the to the of the of venom and an of each were tested for their antibacterial activity against Gram-negative and Gram-positive activity was determined by the in were with 10 μl of the After at were by the peptides were tested against a of Gram-negative and Gram-positive microbial the and fungi in activity was determined by the A of and with was with a of the at and into in were with μl of the were at °C to the of the inhibitory and for at °C or on the activity was also by the The fungal colonies were into the close to a with the and from growth of the fungal colonies after of was as fungal The minimal inhibitory concentration of the peptides and was determined by liquid and growth on the strains in In the growth the were determined by a from an concentration of by in a buffer, pH activity was performed as described In the liquid growth μl of a of bacteria were to μl of growth was in by an in after at °C or The concentrations tested for each peptide were in the of The are as the concentration at which bacteria and the concentration that growth P. C. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). B from the from honey bee venom, and from the of the were as control antibiotic peptides in the growth action of ponericins G1, and are as the the concentration tested at which the bacteria and the that growth growth growth action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested melittin, and were as peptides in the growth are as the the concentration tested at which the bacteria and the that growth action was at the concentration tested in a melittin, and were as peptides in the growth A of was with of or in were with μl of the peptide solution and at °C to the of the hemolytic After at and were of each peptide or a of of each of the 10 peptides was solubilized in an peptide were injected into of cricket 10 or P. of an or a dried solution of acetonitrile and trifluoroacetic acid in a The to the of of the larvae after was by analysis A of the Press, Scholar). The were in a using a The four peptides, ponericins G1, and were solubilized in with and trifluoroethanol at at concentrations of and The were and with a a and spectrum was from The and were the same and then from the The structure was from each spectrum using the analysis P. PubMed Scopus Google Scholar). antibacterial of the venom of P. action against both Gram-positive and Gram-negative strains at a of The venom was separated on a reversed-phase into four of which only an antibacterial activity against the of the peptides in demonstrated that of the collected antibacterial properties The active peptides present in were analyzed by matrix-assisted laser ionization/desorption time-of-flight mass or were further studied. In total, 15 peptides were characterized at the of their amino acid sequences using Edman The amino acid sequence was also possible for peptides present in For some of the analyzed peptides, carboxypeptidase Y were to or of the amino acid. The of the with the was as a control for each sequence peptides can be classified into three families to their primary structures The peptides were named ponericins G, W, or L to the most amino acid. Within each most of the peptides share a of sequence with each to or for ponericins G, W, or The sequence similarities were using and Ponericins and present the sequence with the of the ponericin G and W families to for and and from to for were classified in the families because they some sequence features with the further investigate the antibacterial spectrum and insecticidal activity of each 10 of these 15 peptides to from each were synthesized as and Whole venom was active against the tested microbial strains the fungi Among the the most Gram-positive bacteria were B. B. and the most Gram-negative The 10 peptides four of antibacterial action at a concentration of Ponericins and an action to that of venom and were active against the tested bacteria and the The to the peptides and For the of the growth against Gram-positive bacteria were also to those with venom, but at the tested these peptides were active against most of the Gram-negative Within was the only that not affect the The of peptides and was active against most of the Gram-positive strains but against the Gram-negative with the of P. and Finally, ponericin only a of the tested be that of the peptides were against the fungal as for peptides G1, and were for further analyses. member of each peptide a activity spectrum was for which was active against only a Their were determined in both liquid and growth against Gram-positive and Gram-negative strains or by most of the peptides In the liquid growth G1, and a activity against most of the and were In the growth were at the of peptide in a the same of were with a action of peptide against most of the tested the comparison of their in the growth with those of melittin, and as peptides that their antimicrobial activity or for The hemolytic activity of the venom and the 10 peptides was tested on both and at the same concentration as the antimicrobial activity spectrum the were the most Whole venom and three peptides and of both and and were active and hemolytic activity only against The peptides not exhibit hemolytic activity. The insecticidal properties of P. venom were only in C. 10 peptides insecticidal properties against A. were active against G3, and with Among these four peptides, and were the only that P. the of against ants was a possible of these ants against their action injected of the 10 peptides on the and ant of P. of the 10 action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested action was at the concentration tested in a The concentration of each of the 10 peptides from to in the venom The from the growth in which the tested concentrations of each were and that most of the strains were that the antibacterial peptide in concentration to exhibit a antibacterial activity in the and concentration of the 10 synthesized peptides present in venom of P. of peptide venom of of ponericin was from the comparison of with the of the peptide of peptide present in venom was determined by the of the fractions to each peptide to the of a known of The concentration of each peptide was with a of the venom of by the to an The of ponericin was from the comparison of with the of the peptide in a The of peptide present in venom was determined by the of the fractions to each peptide to the of a known of The concentration of each peptide was with a of the venom of by the to an some structural features of the ponericins with known antibacterial peptides for which extensive structural have been we analyzed the of four peptides in and in The four peptides in as as in The for ponericin are as in In water, the are of a or structure with a nm. The in the of a solvent the of water, the of a high with a at and at and nm. The analysis of the in in a over for four peptides This the on the and of antibacterial peptides from ant 15 peptides, named ponericins and from the venom of the ponerine ant P. goeldii, present sequences not in were classified into three families to their primary structure ponericins G, W, and L. the of peptides, ponericins G, of action can be and have a action against the microbial strains and are also and are only active against some Gram-positive bacteria and and present insecticidal Ponericins G share sequence with using a of antimicrobial peptides that have been isolated from only families, and PubMed Scopus Google Scholar). the of the ponericins G the isolated from that can be as cecropin-like those isolated from C. M. J. S. A. P. J. Biol. 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