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Convergent evolution of invertebrate defensins and nematode antibacterial factors
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TRENDS in Microbiology
Vol.13 No.7 July 2005
Convergent evolution of invertebrate defensins and nematode antibacterial factors Oren Froy Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
Antibacterial factors (ABFs) are secreted polypeptides that have an important role in the innate immune system of nematodes. Comparison of these polypeptides revealed similarity in bioactivity, protein sequence and 3D structure, suggesting that they originated from a common ancestor. Comparison of gene organization of nematode ABF genes revealed that all except one contain a Phase 0 intron at a conserved location. The intron phase and location are congruent with the postulated intron gain rules, suggesting a gain of intron before duplication and divergence of the ancestral gene. Although nematode ABFs display similarity in activity and structure to invertebrate (arthropod and mollusk) defensins, lack of sequence similarity and the different genomic organization suggest that these two polypeptide families exhibit convergent evolution. Innate immunity The immune system provides protection against a wide variety of pathogens. One component of the immune system, the innate immunity, is phylogenetically ancient and is found in plants and animals [1]. The innate immunity confers broad protection against pathogens, and most multicellular organisms depend upon it to combat microbial infections. The innate immunity of vertebrates and invertebrates consists in part of small (35–50 amino acids) cationic antibacterial proteins secreted in response to bacterial or septic injuries [2–5]. Arthropods (insects and arachnids) and mollusks have a plethora of antibacterial polypeptides, among which the defensin family is the most abundant [6]. Nematodes possess five groups of antimicrobial factors that can be classified as either enzymes or peptides and that have been suggested to contribute to immunity. These five groups are lysozymes, caenopores (amoebapore-like enzymes), lipases, glycine-rich putative anti-microbial peptides and antibacterial factors [1]. Thus far, 12 antibacterial factors (ABFs) have been isolated from nematodes (Figure 1), six from the pig roundworm Ascaris suum (ASABF-a, ASABF-b, ASABF-g, ASABF-d, ASABF-3, ASABF-z) and six from the soil nematode Caenorhabditis elegans (ABF-1, ABF-2, ABF-3, ABF-4, Corresponding author: Froy, O. ([email protected]). Available online 23 May 2005
ABF-5, ABF-6) [7–11]. The recent isolation of nematode antibacterial factors and their resemblance to invertebrate (arthropod and mollusk) defensins piqued the interest of researchers to study their evolutionary trajectory. Herein, I compare the amino acid sequence, bioactivity, 3D structure and genomic organization of invertebrate defensins and nematode ABFs. This comparison reveals that these two protein families most likely do not share a common ancestor but have developed via convergent evolution to exhibit a similar antibacterial mode-of-action. Amino acid sequence of invertebrate defensins and nematode ABFs Sequence comparison of full-length nematode ABFs reveals little similarity. However, high homology in the ABFs appears in the region of the mature polypeptide, the region encompassing the eight cysteines (Figure 1). This region reveals 25–95% similarity (Figures 1 and 2). The C-terminal region (C-terminal to the last cysteine) is divergent and varies in length (Figure 1). Most likely, the C-terminal region is cleaved off post-translationally, as was shown for ASABF-a [7,12]. The resemblance in the sequence of the mature region (the region encompassing the eight cysteines; see Figure 1) of nematode ABFs suggests that they all have a similar mode-of-action. Similarly, the high degree of identity among defensins from mollusks and arthropods is found only at the region encompassing the six (insect and arachnid defensins) or eight cysteines (mollusk defensins) [6]. Bioactivity of invertebrate defensins and nematode ABFs Invertebrate (arthropod and mollusk) defensins are induced when the organism is exposed to, or challenged with, bacteria [3,4]. Similarly, injection of heat-killed Escherichia coli OP50 into the pseudocoelom of A. suum showed that ASABF transcripts were upregulated in the body wall and intestine [9]. When induced, ABFs kill bacteria rapidly because less than 1 min of contact is enough for ASABF-a to kill Staphylococcus aureus [7,8,12]. Thus far, the bioactivities of only ASABF-a and ABF-2 have been characterized. ASABF-a and ABF-2 are active against the Gram-positive bacteria Bacillus subtilis, Clavibacter michiganensis pv. Michiganensis, Curtobacterium flaccumfaciens pv. ooritt, Kocuria varians,
www.sciencedirect.com 0966-842X/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2005.05.001
Opinion
TRENDS in Microbiology
Vol.13 No.7 July 2005
ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ε ASABF-ζ ABF-1 ABF-2 ABF-3 ABF-4 ABF-5 ABF-6
* * * * * * * * * * MKT--AIIVVLLVIFASTNAAVDFSSCA--RMDVPGL-SKVAQGLCISSCKFQNCGTGHCEKR---GGRPTCVCDRC MKT--IVVVILFVAVVAVTSAIDFSSCA--RMDVPGL-NKVAQGLCISSCKYQNCGTGHCEKR---GGRPTCVCGRC MKT--IIFTVLFVAVVAVTSAIDFSTCA--RMDVPGL-SKVAQGSCISSCKFQNCGTGHCEKR---GGRPTCVCSRC MRI--FTAVVLIVVLVCVNAGIDFSTCA--RMDVPGL-SKVARGLCINSCKFQNCGTGYCEKR---GGRPTCVCSRC MVTKGIVLFMLVILFASTDAA----TCG---YDDAKL-NRPTIG-CILSCKVQGCETGACYLR---DSRPICVCKRC MKA--ILIALLLTTFTVVNGGVVLTSCA--RMDTPVL-SKAAQGLCITSCKYQNCGTGFCQKV---GGRPTCMCRRC MLY----FCLLLVLLLPNNGVSSEASCA--RMDVPVM-QRIAQGLCTSSCTAQKCMTGICKKV---DSHPTCFCGGC MFVRSLFLALLLATIVA--ADIDFSTCA--RMDVPIL-KKAAQGLCITSCSMQNCGTGSCKKR---SGRPTCVCYRC MNF--SFLFFIFAFLIGLNKG---SVCLTRRTDWGQLGAIFTNPVCDVWCRIRQCGPGQCKEDPMTSDEAQCVCEKC MIC-NCFLLIIVTLVISNCDGI----CL-NHEGWGNVGSVFTDPLCNVWCEIKLCGPGQCIEDPMTTAPARCVCEKC MNYNFLLLSACIIFLIPEKSE---SICVTRRTDWGQFGSFFTDPLCDVWCRIRRCGKGQCREDPATSNTANCVCEKC MFR-KLIIATFVLSLCDLANSV--TICS-----SSSLLSTFTDPLCTSWCKVRFCSSGSCRSV-MSGSDPTCECESC
ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ε ASABF-ζ ABF-1 ABF-2 ABF-3 ABF-4 ABF-5 ABF-6
-----------------------GRGGG---EWPSVPMPKG-RSSRGRRHS------------------------------------------------GTGGG---EWPSVPMPKGGKKGR-----------------------------------------------------GSGGG---SWPSVGK-------------------------------------------------------------DNGGG---SWP--GRK----------------------------------------------------------------------------------------------------------------------------------------ANGGG---SWPVIPLDTLVKLALKRGKR--------------------------------------SN--------ANDVSLDTLISQLPHN------------------------------------------------------------ANGGG------DIPLGALIKRG----------------------------------------------------YRDSYGN-----AIYPGNNGLQ------------------------------------------------------YRDNSGN-----AVYPGGNNDYQQQAQMNRERQLDR-QYNRQLRQQNRMNKAWQRG ---------------------YRDDDGN-----VIFPDNDGFQQSRLNFDNSPTSSS-PWTMNQRNEDDLYPSQDRY GFGSWFGSSSDSNSNQPVSGQYYAGGSGGEMATPNYGNNNGYNNGYNNGNNMRYNDNNGYNTNNGYRGQPTPGYGNS
ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ε ASABF-ζ ABF-1 ABF-2 ABF-3 ABF-4 ABF-5 ABF-6
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------WQ-----------------------------------------------DTDRN--------------------------------------------NSNFNSNQQYSYQQYYNNRNNQYGNSGYGNAGQAGQTGYPSGYQNLKKKR
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Figure 1. Sequence alignment of nematode antibacterial factors. Asterisks designate identical residues in all proteins, cysteine residues are in red and identical non-cysteine residues are in yellow. The blue triangle represents the location of the intron in all the genes encoding ABFs except that of ABF-2. ABF sequences were analyzed using the ClustalW 1.75 program. ASABF-a, accession number BAA89497; ASABF-b, accession number BAC00497; ASABF-g, accession number BAC00498; ASABF-d, accession numbers BAC00499; ASABF-3, accession number BAC41495; ASABF-z, accession number BAC57992; ABF-1, accession number AAK68260; ABF-2, accession number AAK68258; ABF-3, accession number NP_506950; ABF-4, accession number NP_507965; ABF-5, accession number NP_510136; ABF-6, accession number NP_741914.
Staphylococcus aureus and Staphylococcus saprophyticus at minimum bactericidal concentration (MBC) 0.03–1 mg mlK1. These ABFs were also found to be active against the Gram-negative bacteria Agrobacterium tumefaciens, Klebsiella pneumoniae, Bdellovibrio bacteriovorus and Erwinia carotovora subsp. Carotovora at MBC O0.5 mg mlK1. ASABF-a and ABF-2 are also active against some yeasts, such as Kluyveromyces thermotolerans, Candida krusei and Pichia anomala at MBC O3 mg mlK1. Thus, ASABF-a and ABF-2 exhibit potent antibacterial activity against Gram-positive bacteria, weaker activity against some Gram-negative bacteria and even weaker activity against some yeast species. Slight hemolytic activity (4.2% at 100 mg mlK1) against human erythrocytes was also observed but only under low ionic strength conditions [7,8,12]. Similar to nematode ABFs, arthropod and mollusk defensins have also been shown to have antimicrobial activity against a wide range of Gram-positive and a few Gram-negative bacteria [13]. In addition, defensins have been shown to disrupt the www.sciencedirect.com
bacterial membrane by interacting with membrane phospholipids and forming complexes that are not miscible in the lipid phase [14–16]. Structure of invertebrate defensins and nematode ABFs The determined structures of invertebrate defensins reveal that, regardless of their length and number of S–S bonds, they consist of an a-helix and two antiparallel b-strands stabilized by three (Defensin A) or four (MGD-1) disulfide bonds constituting the common cysteinestabilized a/b motif (Figure 3). The cysteine-stabilized a/b (CSa/b) motif involves a Cys-Xaa-Xaa-Xaa-Cys stretch of the a-helix bonded through two disulfide bridges to a Cys-Xaa-Cys triplet of a b-strand [6,17–21]. This motif is also found in a wide range of membrane potential modulators, such as scorpion neurotoxins, snake sarafotoxins and plant g-thionins [6,17,22,23]. Mature ASABF-a contains four intra-molecular disulfide bridges [7]. NMR determination of ASABF-a revealed that it also contains a cysteine-stabilized a/b (CSa/b) motif [24]. Sequence
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ASABF-α
Glossary
ASABF-β
Intron phase: An intron is a DNA segment of a transcribed gene that is removed in the process of RNA splicing and does not appear in the mature mRNA molecule. Introns reside between exons and their location could be one of three phases: Phase 0 intron – an intron that lies between two codons. Phase 1 intron – an intron that lies between the first and second nucleotides of a codon. Phase 2 intron – an intron that lies between the second and third nucleotides of a codon. Intron gain: The phenomenon of random intron addition into genes during eukaryotic evolution. Exon shuffling: Exon shuffling is proposed to have occurred because the same or highly similar exons appear in otherwise non-related genes, which are located in different genomic regions. Exon shuffling is the process by which exons are duplicated in either the same DNA molecule, or by which structural or functional domains are exchanged among genes. Exon shuffling is considered to have contributed to the genetic diversity and complexity that is the hallmark of vertebrate and invertebrate genomes. Walter Gilbert put forward the hypothesis that exons code for functional units of a protein and that the evolution of new genes was preceded by recombination or exclusion of exons [35]. Convergent evolution: Convergent evolution is the emergence of biological structures or species that exhibit similar function and appearance but that evolved through widely divergent evolutionary pathways. When homologous features become used for different purposes, the process is called ‘divergent evolution’ – the splitting of a family tree in different directions. When unrelated groups produce similar structures, even if they are based on different origin (wings in bats and flies), the process is called ‘convergent evolution’.
ASABF-γ ASABF-δ ASABF-ζ ABF-2 ABF-1 ASABF-ε ABF-3 ABF-5 ABF-4 ABF-6 TRENDS in Microbiology
Figure 2. Phylogenetic tree of nematode ABFs. The protein regions taken for analysis were the leader peptide and the region encoding the CSa/b region until the last cysteine. ASABF-a, accession number BAA89497; ASABF-b, accession number BAC00497; ASABF-g, accession number BAC00498; ASABF-d, accession numbers BAC00499; ASABF-3, accession number BAC41495; ASABF-z, accession number BAC57992; ABF-1, accession number AAK68260; ABF-2, accession number AAK68258; ABF-3, accession number NP_506950; ABF-4, accession number NP_507965; ABF-5, accession number NP_510136; ABF-6, accession number NP_741914.
similarity (Figure 1) and the fact that all other members of nematode ABFs have eight cysteine residues imply that they all contain four disulfide bridges and a cysteinestabilized a/b motif. Genomic organization of invertebrate defensins and nematode ABFs ABF-1 and ABF-2 are located on chromosome I, ABF-3 and ABF-4 on chromosome V, and ABF-5 and ABF-6 on chromosome X. All ABFs except ABF-2 have an intron at a conserved location, splitting the region encoding the CSa/b motif [8,9] (Figures 1 and 3). Although ABF-2 shares 57% identity at the amino acid level of the CSa/b region with ABF-1, it harbors no intron in the gene. Nematode genes have a particularly high rate of intron gain (see Glossary) and loss compared with other animals [25], therefore, the introns in the ABF genes vary in length (Figure 3) but they are all of Phase 0. The sequence in which the intron is located (Y) is MYYYV, in which M is either A or C, Y is either A or G, and V is either A, G or C. This consensus sequence is similar to that postulated for intron gain, MAGYR, in which M is either A or C and R is either C or T [26–28]. In contrast to nematode ABFs, arthropod and mollusk defensin genes exhibit diverse number and location of introns, all introns are of Phase I and the region encoding the CSa/b motif is never split by an intron [6]. Evolution of nematode ABFs Sequence identity (Figure 1), bioactivity, structural commonality (Figure 3) and genomic organization (Figure 3) clearly indicate that nematode ABFs are genetically related [9] (Figure 2). The intron was presumably gained into the ABF gene after initial duplication www.sciencedirect.com
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of the gene because ABF-2 does not contain an intron. After intron gain, the gene was further duplicated and underwent natural mutagenesis to generate the extant different variants in both A. suum and C. elegans. Similar to nematode ABFs, comparison of mollusk and arthropod defensin proteins also showed that they exhibit commonality at three levels: sequence identity, bioactivity and 3D structure [6]. However, gene analysis of mollusk and arthropod defensins revealed that: (1) the exon encoding the mature defensin is not split by an intron; (2) the introns flanking the exon encoding the mature defensin are of the same phase, Phase I; and (3) the mature defensin encoded by this exon displays folding autonomy as one structural entity. These findings fulfilled the criteria postulated to determine exon shuffling in other genes [29,30] and led us to suggest that exon shuffling occurred in the exon encoding the mature defensin during evolution [6]. ABF gene organization is in contrast to the criteria postulated for exon shuffling [29–31]: (1) the structural entity is split by an intron and (2) the gene contains a Phase 0 intron suggesting intron gain. The criteria of intron gain alongside different genomic organization and lack of sequence similarity suggest that, although invertebrate defensins and nematode ABFs exhibit similarity in bioactivity and structure, they most likely developed through convergent evolution. This notion is further substantiated by a phylogenetic analysis showing that these two polypeptide groups have developed independently (Figure 4). It is also possible that over millions of years of evolution, ABF genes have been exposed to strong selection pressures gaining mutations that assist in combating nematode-specific pathogens. Also, the high incidence of intron gain [25] and different location of introns in nematode genes compared with those of other organisms [32] could account for the different genomic organization between nematode ABFs and invertebrate defensins. Although theoretically possible, common ancestry of nematode ABFs and invertebrate
Opinion
TRENDS in Microbiology
Vol.13 No.7 July 2005
MGD-1
L
CSα/β
Intron
ASABF-α
1496 bp
ASABF-β
295 bp
ASABF-γ
295 bp
ASABF-δ
>202 bp
ASABF-ε
CSα/β
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Defensin A
C terminal
>8 bp
ASABF-ζ
>69 bp
ABF-1
712 bp
ABF-2 ABF-3
167 bp
ABF-4
54 bp
ABF-5 ABF-6
54 bp 51 bp
Figure 3. Schematic gene organization of nematode ABFs. The coding region for the leader peptide (L) appears in black. The region encoding the CSa/b and C-terminal regions are grey and hatched, respectively. The intron is depicted as a white bar and its size in base pairs is designated. The relative size of all gene regions, except for that of the intron, is drawn to scale. ASABF-a, accession number AB029816; ASABF-b, accession number AB029812; ASABF-g, accession number AB029813; ASABF-d, accession numbers AB029814, AB029815; ASABF-3, accession number AB090347S1; ASABF-z, accession number AB090349S1; ABF-1 and ABF-2, accession number NC_003279; ABF-3 and ABF-4, accession number NC_003283; ABF-5 and ABF-6, accession number NC_003284. Inset: the cysteine-stabilized a/beta structural motif common to all arthropod and mollusk defensins. The structure of the Mediterranean mussel Mytilus galloprovincialis defensin 1 (MGD-1; left) and the fly Protophormia terraenovae Defensin A (right) (PDB entries 1FJN and 1ICA, respectively) are presented. Alpha helices are presented in green; b strands in blue. The models were generated using the RasMol Version 2.6 software.
defensins would have had to involve a large number of events. These events would have had to include losing the introns flanking the shuffling exon, gaining an intron in the middle of the exon encoding the mature ABFs, and gaining a large number of mutations. Therefore, although still putative, convergent evolution seems a more feasible trajectory for the development of nematode ABFs and invertebrate defensins. All reported CSa/b-containing antibacterial polypeptides are translated as precursors containing a signal peptide at their N terminus to ensure secretion. Some CSa/b-containing antimicrobial peptides also contain a pro-region, which is removed post-translationally to generate mature peptides before secretion. The pro-region is usually located between the signal peptide and the mature region in some CSa/b-containing antimicrobial peptides (insect defensins) [6]. In ABFs, the secretory signal is located N-terminal to the mature region and the pro-region is C-terminal to the mature peptide. This organization, although different than that of insect defensins, is similar to that of MGD-1 and myticin A, www.sciencedirect.com
two mollusk defensins [11,33]. This finding instigated Zhang and Kato [11] to suggest that nematode ABFs and mollusk MGD-1 and myticin A are genetically related. In addition, comparison of ASABF-a, ABF-4, ABF-5 and ABF-6 to other CSa/b-containing polypeptides supported the hypothesis raised by Zhang and Kato [11] because gene organization and the cysteine array of nematode ABFs are similar to that of the mollusk defensin MGD-1 and myticin A [34]. Recently, we suggested that the exon encoding the mature MGD-1 is genetically related to mature arthropod defensins because they share high similarity in sequence, structure, bioactivity and genomic organization [6]. In addition, as described earlier, invertebrate (arthropod and mollusk) defensins and nematode ABFs developed independently throughout evolution (Figure 4). Therefore, it is unlikely that MGD-1 and nematode ABFs are genetically related. Although proposed by Zhang and Kato [11], the authors also add that there is no highly reliable evidence, such as sequence identity or conservative genomic organization, for common ancestry of MGD-1 and ABFs.
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Dm Aa-B Lq Om-A MGD-1 ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ζ ABF-2 ABF-1 ASABF-ε ABF-3 ABF-5 ABF-4 ABF-6 Figure 4. Phylogenetic tree of invertebrate defensins and nematode ABFs. The protein regions taken for analysis were the regions encoding the CSa/b. ASABF-a, accession number BAA89497; ASABF-b, accession number BAC00497; ASABF-g, accession number BAC00498; ASABF-d, accession numbers BAC00499; ASABF-3, accession number BAC41495; ASABF-z, accession number BAC57992; ABF-1, accession number AAK68260; ABF-2, accession number AAK68258; ABF-3, accession number NP_506950; ABF-4, accession number NP_507965; ABF-5, accession number NP_510136; ABF-6, accession number NP_741914. Aa-B, Aedes aegypti defensin B (accession number AAB35030); Dm, Drosophila melanogaster (accession number S43228); Lq, Leiurus quinquestriatus (accession number JN0613); MGD-1, Mytilus galloprovincialis defensin 1 (accession number S74088); Om-A, Ornithodoros moubata defensin A (accession number BAB41028).
Concluding remarks Sequence identity, bioactivity, structural commonality and genomic organization revealed that nematode ABFs are genetically related [9]. ABF gene analysis revealed that they might have gained an intron along evolution, before duplication and diversification of the genes. Recently, we have suggested that invertebrate (arthropod and mollusk) defensins are genetically related and have most likely evolved via exon shuffling [6,17]. Although nematode ABFs display similarity in activity and structure to invertebrate defensins, lack of sequence similarity and the different genomic organization suggest that these two polypeptide families exhibit an example of convergent evolution. To have a general picture of the evolutionary trajectory of antibacterial polypeptides, researchers must invest more time to isolate and analyze new members from other phyla. Comparative analysis of the genomic organization of a large number of genes will enable us to determine whether they exhibit common ancestry or convergent evolution. Thus, future isolation and analysis of novel genes encoding antibacterial polypeptides could hold the key to the mysterious phylogenetic trajectory of these unique molecules.
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5 Mallo, G.V. et al. (2002) Inducible antibacterial defense system in C. elegans. Curr. Biol. 12, 1209–1214 6 Froy, O. and Gurevitz, M. (2003) Arthropod and mollusk defensins – evolution by exon-shuffling. Trends Genet. 19, 684–687 7 Kato, Y. and Komatsu, S. (1996) ASABF, a novel cysteine-rich antibacterial peptide isolated from the nematode Ascaris suum. Purification, primary structure, and molecular cloning of cDNA. J. Biol. Chem. 271, 30493–30498 8 Kato, Y. et al. (2002) abf-1 and abf-2, ASABF-type antimicrobial peptide genes in Caenorhabditis elegans. Biochem. J. 361, 221–230 9 Pillai, A. et al. (2003) Induction of ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides by bacterial injection: novel members of ASABF in the nematode Ascaris suum. Biochem. J. 371, 663–668 10 Andersson, M. et al. (2003) Ascaris nematodes from pig and human make three antibacterial peptides: isolation of cecropin P1 and two ASABF peptides. Cell. Mol. Life Sci. 60, 599–606 11 Zhang, H. and Kato, Y. (2003) Common structural properties specifically found in the CSalphabeta-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? Dev. Comp. Immunol. 27, 499–503 12 Zhang, H. et al. (2000) In vitro antimicrobial properties of recombinant ASABF, an antimicrobial peptide isolated from the nematode Ascaris suum. Antimicrob. Agents Chemother. 44, 2701–2705 13 Otvos, L., Jr. (2000) Antibacterial peptides isolated from insects. J. Pept. Sci. 6, 497–511 14 Matsuyama, K. and Natori, S. (1990) Mode of action of sapecin, a novel antibacterial protein of Sarcophaga peregrina (flesh fly). J. Biochem. (Tokyo) 108, 128–132 15 Cociancich, S. et al. (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268, 19239–19245 16 Maget-Dana, R. and Ptak, M. (1997) Penetration of the insect defensin A into phospholipid monolayers and formation of defensin A-lipid complexes. Biophys. J. 73, 2527–2533 17 Froy, O. and Gurevitz, M. (1998) Membrane potential modulators: a thread of scarlet from plants to humans. FASEB J. 12, 1793–1796 18 Hanzawa, H. et al. (1990) 1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin. FEBS Lett. 269, 413–420 19 Bonmatin, J.M. et al. (1992) Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding. J. Biomol. NMR 2, 235–256 20 Cornet, B. et al. (1995) Refined three-dimensional solution structure of insect defensin A. Structure 3, 435–448 21 Yang, Y.S. et al. (2000) Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1). Biochemistry 39, 14436–14447 22 Froy, O. and Gurevitz, M. (2003) New insight on scorpion divergence inferred from comparative analysis of toxin structure, pharmacology, and distribution. Toxicon 42, 549–555 23 Froy, O. and Gurevitz, M. (2004) Arthropod defensins illuminate the divergence of scorpion neurotoxins. J. Pept. Sci. 10, 714–718 24 Aizawa, T. et al. (2000) Structural analysis of an antibacterial peptide derived from a nematode. In Peptide Science (Shioiri, T., ed.), pp. 269–272, The Japanese Peptide Society 25 Logsdon, J.M., Jr. et al. (1995) Seven newly discovered intron positions in the triose-phosphate isomerase gene: evidence for the introns-late theory. Proc. Natl. Acad. Sci. U. S. A. 92, 8507–8511 26 Logsdon, J.M., Jr. (2004) Worm genomes hold the smoking guns of intron gain. Proc. Natl. Acad. Sci. U. S. A. 101, 11195–11196 27 Coghlan, A. and Wolfe, K.H. (2004) Origins of recently gained introns in Caenorhabditis. Proc. Natl. Acad. Sci. U. S. A. 101, 11362–11367 28 Qiu, W.G. et al. (2004) The evolutionary gain of spliceosomal introns: sequence and phase preferences. Mol. Biol. Evol. 21, 1252–1263 29 Patthy, L. (1994) Introns and exons. Curr. Opin. Struct. Biol. 4, 383–392 30 Patthy, L. (1999) Genome evolution and the evolution of exon-shuffling – a review. Gene 238, 103–114 31 Ponting, C.P. and Russell, R.R. (2002) The natural history of protein domains. Annu. Rev. Biophys. Biomol. Struct. 31, 45–71
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32 Gotoh, O. (1998) Divergent structures of Caenorhabditis elegans cytochrome P450 genes suggest the frequent loss and gain of introns during the evolution of nematodes. Mol. Biol. Evol. 15, 1447–1459 33 Mitta, G. et al. (2000) Original involvement of antimicrobial peptides in mussel innate immunity. FEBS Lett. 486, 185–190
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34 Mitta, G. et al. (1999) Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis. Eur. J. Biochem. 265, 71–78 35 Dorit, R.L. and Gilbert, W. (1991) The limited universe of exons. Curr. Opin. Genet. Dev. 1, 464–469
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Convergent evolution of invertebrate defensins and nematode antibacterial factors Oren Froy Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
Antibacterial factors (ABFs) are secreted polypeptides that have an important role in the innate immune system of nematodes. Comparison of these polypeptides revealed similarity in bioactivity, protein sequence and 3D structure, suggesting that they originated from a common ancestor. Comparison of gene organization of nematode ABF genes revealed that all except one contain a Phase 0 intron at a conserved location. The intron phase and location are congruent with the postulated intron gain rules, suggesting a gain of intron before duplication and divergence of the ancestral gene. Although nematode ABFs display similarity in activity and structure to invertebrate (arthropod and mollusk) defensins, lack of sequence similarity and the different genomic organization suggest that these two polypeptide families exhibit convergent evolution. Innate immunity The immune system provides protection against a wide variety of pathogens. One component of the immune system, the innate immunity, is phylogenetically ancient and is found in plants and animals [1]. The innate immunity confers broad protection against pathogens, and most multicellular organisms depend upon it to combat microbial infections. The innate immunity of vertebrates and invertebrates consists in part of small (35–50 amino acids) cationic antibacterial proteins secreted in response to bacterial or septic injuries [2–5]. Arthropods (insects and arachnids) and mollusks have a plethora of antibacterial polypeptides, among which the defensin family is the most abundant [6]. Nematodes possess five groups of antimicrobial factors that can be classified as either enzymes or peptides and that have been suggested to contribute to immunity. These five groups are lysozymes, caenopores (amoebapore-like enzymes), lipases, glycine-rich putative anti-microbial peptides and antibacterial factors [1]. Thus far, 12 antibacterial factors (ABFs) have been isolated from nematodes (Figure 1), six from the pig roundworm Ascaris suum (ASABF-a, ASABF-b, ASABF-g, ASABF-d, ASABF-3, ASABF-z) and six from the soil nematode Caenorhabditis elegans (ABF-1, ABF-2, ABF-3, ABF-4, Corresponding author: Froy, O. ([email protected]). Available online 23 May 2005
ABF-5, ABF-6) [7–11]. The recent isolation of nematode antibacterial factors and their resemblance to invertebrate (arthropod and mollusk) defensins piqued the interest of researchers to study their evolutionary trajectory. Herein, I compare the amino acid sequence, bioactivity, 3D structure and genomic organization of invertebrate defensins and nematode ABFs. This comparison reveals that these two protein families most likely do not share a common ancestor but have developed via convergent evolution to exhibit a similar antibacterial mode-of-action. Amino acid sequence of invertebrate defensins and nematode ABFs Sequence comparison of full-length nematode ABFs reveals little similarity. However, high homology in the ABFs appears in the region of the mature polypeptide, the region encompassing the eight cysteines (Figure 1). This region reveals 25–95% similarity (Figures 1 and 2). The C-terminal region (C-terminal to the last cysteine) is divergent and varies in length (Figure 1). Most likely, the C-terminal region is cleaved off post-translationally, as was shown for ASABF-a [7,12]. The resemblance in the sequence of the mature region (the region encompassing the eight cysteines; see Figure 1) of nematode ABFs suggests that they all have a similar mode-of-action. Similarly, the high degree of identity among defensins from mollusks and arthropods is found only at the region encompassing the six (insect and arachnid defensins) or eight cysteines (mollusk defensins) [6]. Bioactivity of invertebrate defensins and nematode ABFs Invertebrate (arthropod and mollusk) defensins are induced when the organism is exposed to, or challenged with, bacteria [3,4]. Similarly, injection of heat-killed Escherichia coli OP50 into the pseudocoelom of A. suum showed that ASABF transcripts were upregulated in the body wall and intestine [9]. When induced, ABFs kill bacteria rapidly because less than 1 min of contact is enough for ASABF-a to kill Staphylococcus aureus [7,8,12]. Thus far, the bioactivities of only ASABF-a and ABF-2 have been characterized. ASABF-a and ABF-2 are active against the Gram-positive bacteria Bacillus subtilis, Clavibacter michiganensis pv. Michiganensis, Curtobacterium flaccumfaciens pv. ooritt, Kocuria varians,
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ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ε ASABF-ζ ABF-1 ABF-2 ABF-3 ABF-4 ABF-5 ABF-6
* * * * * * * * * * MKT--AIIVVLLVIFASTNAAVDFSSCA--RMDVPGL-SKVAQGLCISSCKFQNCGTGHCEKR---GGRPTCVCDRC MKT--IVVVILFVAVVAVTSAIDFSSCA--RMDVPGL-NKVAQGLCISSCKYQNCGTGHCEKR---GGRPTCVCGRC MKT--IIFTVLFVAVVAVTSAIDFSTCA--RMDVPGL-SKVAQGSCISSCKFQNCGTGHCEKR---GGRPTCVCSRC MRI--FTAVVLIVVLVCVNAGIDFSTCA--RMDVPGL-SKVARGLCINSCKFQNCGTGYCEKR---GGRPTCVCSRC MVTKGIVLFMLVILFASTDAA----TCG---YDDAKL-NRPTIG-CILSCKVQGCETGACYLR---DSRPICVCKRC MKA--ILIALLLTTFTVVNGGVVLTSCA--RMDTPVL-SKAAQGLCITSCKYQNCGTGFCQKV---GGRPTCMCRRC MLY----FCLLLVLLLPNNGVSSEASCA--RMDVPVM-QRIAQGLCTSSCTAQKCMTGICKKV---DSHPTCFCGGC MFVRSLFLALLLATIVA--ADIDFSTCA--RMDVPIL-KKAAQGLCITSCSMQNCGTGSCKKR---SGRPTCVCYRC MNF--SFLFFIFAFLIGLNKG---SVCLTRRTDWGQLGAIFTNPVCDVWCRIRQCGPGQCKEDPMTSDEAQCVCEKC MIC-NCFLLIIVTLVISNCDGI----CL-NHEGWGNVGSVFTDPLCNVWCEIKLCGPGQCIEDPMTTAPARCVCEKC MNYNFLLLSACIIFLIPEKSE---SICVTRRTDWGQFGSFFTDPLCDVWCRIRRCGKGQCREDPATSNTANCVCEKC MFR-KLIIATFVLSLCDLANSV--TICS-----SSSLLSTFTDPLCTSWCKVRFCSSGSCRSV-MSGSDPTCECESC
ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ε ASABF-ζ ABF-1 ABF-2 ABF-3 ABF-4 ABF-5 ABF-6
-----------------------GRGGG---EWPSVPMPKG-RSSRGRRHS------------------------------------------------GTGGG---EWPSVPMPKGGKKGR-----------------------------------------------------GSGGG---SWPSVGK-------------------------------------------------------------DNGGG---SWP--GRK----------------------------------------------------------------------------------------------------------------------------------------ANGGG---SWPVIPLDTLVKLALKRGKR--------------------------------------SN--------ANDVSLDTLISQLPHN------------------------------------------------------------ANGGG------DIPLGALIKRG----------------------------------------------------YRDSYGN-----AIYPGNNGLQ------------------------------------------------------YRDNSGN-----AVYPGGNNDYQQQAQMNRERQLDR-QYNRQLRQQNRMNKAWQRG ---------------------YRDDDGN-----VIFPDNDGFQQSRLNFDNSPTSSS-PWTMNQRNEDDLYPSQDRY GFGSWFGSSSDSNSNQPVSGQYYAGGSGGEMATPNYGNNNGYNNGYNNGNNMRYNDNNGYNTNNGYRGQPTPGYGNS
ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ε ASABF-ζ ABF-1 ABF-2 ABF-3 ABF-4 ABF-5 ABF-6
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------WQ-----------------------------------------------DTDRN--------------------------------------------NSNFNSNQQYSYQQYYNNRNNQYGNSGYGNAGQAGQTGYPSGYQNLKKKR
315
Figure 1. Sequence alignment of nematode antibacterial factors. Asterisks designate identical residues in all proteins, cysteine residues are in red and identical non-cysteine residues are in yellow. The blue triangle represents the location of the intron in all the genes encoding ABFs except that of ABF-2. ABF sequences were analyzed using the ClustalW 1.75 program. ASABF-a, accession number BAA89497; ASABF-b, accession number BAC00497; ASABF-g, accession number BAC00498; ASABF-d, accession numbers BAC00499; ASABF-3, accession number BAC41495; ASABF-z, accession number BAC57992; ABF-1, accession number AAK68260; ABF-2, accession number AAK68258; ABF-3, accession number NP_506950; ABF-4, accession number NP_507965; ABF-5, accession number NP_510136; ABF-6, accession number NP_741914.
Staphylococcus aureus and Staphylococcus saprophyticus at minimum bactericidal concentration (MBC) 0.03–1 mg mlK1. These ABFs were also found to be active against the Gram-negative bacteria Agrobacterium tumefaciens, Klebsiella pneumoniae, Bdellovibrio bacteriovorus and Erwinia carotovora subsp. Carotovora at MBC O0.5 mg mlK1. ASABF-a and ABF-2 are also active against some yeasts, such as Kluyveromyces thermotolerans, Candida krusei and Pichia anomala at MBC O3 mg mlK1. Thus, ASABF-a and ABF-2 exhibit potent antibacterial activity against Gram-positive bacteria, weaker activity against some Gram-negative bacteria and even weaker activity against some yeast species. Slight hemolytic activity (4.2% at 100 mg mlK1) against human erythrocytes was also observed but only under low ionic strength conditions [7,8,12]. Similar to nematode ABFs, arthropod and mollusk defensins have also been shown to have antimicrobial activity against a wide range of Gram-positive and a few Gram-negative bacteria [13]. In addition, defensins have been shown to disrupt the www.sciencedirect.com
bacterial membrane by interacting with membrane phospholipids and forming complexes that are not miscible in the lipid phase [14–16]. Structure of invertebrate defensins and nematode ABFs The determined structures of invertebrate defensins reveal that, regardless of their length and number of S–S bonds, they consist of an a-helix and two antiparallel b-strands stabilized by three (Defensin A) or four (MGD-1) disulfide bonds constituting the common cysteinestabilized a/b motif (Figure 3). The cysteine-stabilized a/b (CSa/b) motif involves a Cys-Xaa-Xaa-Xaa-Cys stretch of the a-helix bonded through two disulfide bridges to a Cys-Xaa-Cys triplet of a b-strand [6,17–21]. This motif is also found in a wide range of membrane potential modulators, such as scorpion neurotoxins, snake sarafotoxins and plant g-thionins [6,17,22,23]. Mature ASABF-a contains four intra-molecular disulfide bridges [7]. NMR determination of ASABF-a revealed that it also contains a cysteine-stabilized a/b (CSa/b) motif [24]. Sequence
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ASABF-α
Glossary
ASABF-β
Intron phase: An intron is a DNA segment of a transcribed gene that is removed in the process of RNA splicing and does not appear in the mature mRNA molecule. Introns reside between exons and their location could be one of three phases: Phase 0 intron – an intron that lies between two codons. Phase 1 intron – an intron that lies between the first and second nucleotides of a codon. Phase 2 intron – an intron that lies between the second and third nucleotides of a codon. Intron gain: The phenomenon of random intron addition into genes during eukaryotic evolution. Exon shuffling: Exon shuffling is proposed to have occurred because the same or highly similar exons appear in otherwise non-related genes, which are located in different genomic regions. Exon shuffling is the process by which exons are duplicated in either the same DNA molecule, or by which structural or functional domains are exchanged among genes. Exon shuffling is considered to have contributed to the genetic diversity and complexity that is the hallmark of vertebrate and invertebrate genomes. Walter Gilbert put forward the hypothesis that exons code for functional units of a protein and that the evolution of new genes was preceded by recombination or exclusion of exons [35]. Convergent evolution: Convergent evolution is the emergence of biological structures or species that exhibit similar function and appearance but that evolved through widely divergent evolutionary pathways. When homologous features become used for different purposes, the process is called ‘divergent evolution’ – the splitting of a family tree in different directions. When unrelated groups produce similar structures, even if they are based on different origin (wings in bats and flies), the process is called ‘convergent evolution’.
ASABF-γ ASABF-δ ASABF-ζ ABF-2 ABF-1 ASABF-ε ABF-3 ABF-5 ABF-4 ABF-6 TRENDS in Microbiology
Figure 2. Phylogenetic tree of nematode ABFs. The protein regions taken for analysis were the leader peptide and the region encoding the CSa/b region until the last cysteine. ASABF-a, accession number BAA89497; ASABF-b, accession number BAC00497; ASABF-g, accession number BAC00498; ASABF-d, accession numbers BAC00499; ASABF-3, accession number BAC41495; ASABF-z, accession number BAC57992; ABF-1, accession number AAK68260; ABF-2, accession number AAK68258; ABF-3, accession number NP_506950; ABF-4, accession number NP_507965; ABF-5, accession number NP_510136; ABF-6, accession number NP_741914.
similarity (Figure 1) and the fact that all other members of nematode ABFs have eight cysteine residues imply that they all contain four disulfide bridges and a cysteinestabilized a/b motif. Genomic organization of invertebrate defensins and nematode ABFs ABF-1 and ABF-2 are located on chromosome I, ABF-3 and ABF-4 on chromosome V, and ABF-5 and ABF-6 on chromosome X. All ABFs except ABF-2 have an intron at a conserved location, splitting the region encoding the CSa/b motif [8,9] (Figures 1 and 3). Although ABF-2 shares 57% identity at the amino acid level of the CSa/b region with ABF-1, it harbors no intron in the gene. Nematode genes have a particularly high rate of intron gain (see Glossary) and loss compared with other animals [25], therefore, the introns in the ABF genes vary in length (Figure 3) but they are all of Phase 0. The sequence in which the intron is located (Y) is MYYYV, in which M is either A or C, Y is either A or G, and V is either A, G or C. This consensus sequence is similar to that postulated for intron gain, MAGYR, in which M is either A or C and R is either C or T [26–28]. In contrast to nematode ABFs, arthropod and mollusk defensin genes exhibit diverse number and location of introns, all introns are of Phase I and the region encoding the CSa/b motif is never split by an intron [6]. Evolution of nematode ABFs Sequence identity (Figure 1), bioactivity, structural commonality (Figure 3) and genomic organization (Figure 3) clearly indicate that nematode ABFs are genetically related [9] (Figure 2). The intron was presumably gained into the ABF gene after initial duplication www.sciencedirect.com
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of the gene because ABF-2 does not contain an intron. After intron gain, the gene was further duplicated and underwent natural mutagenesis to generate the extant different variants in both A. suum and C. elegans. Similar to nematode ABFs, comparison of mollusk and arthropod defensin proteins also showed that they exhibit commonality at three levels: sequence identity, bioactivity and 3D structure [6]. However, gene analysis of mollusk and arthropod defensins revealed that: (1) the exon encoding the mature defensin is not split by an intron; (2) the introns flanking the exon encoding the mature defensin are of the same phase, Phase I; and (3) the mature defensin encoded by this exon displays folding autonomy as one structural entity. These findings fulfilled the criteria postulated to determine exon shuffling in other genes [29,30] and led us to suggest that exon shuffling occurred in the exon encoding the mature defensin during evolution [6]. ABF gene organization is in contrast to the criteria postulated for exon shuffling [29–31]: (1) the structural entity is split by an intron and (2) the gene contains a Phase 0 intron suggesting intron gain. The criteria of intron gain alongside different genomic organization and lack of sequence similarity suggest that, although invertebrate defensins and nematode ABFs exhibit similarity in bioactivity and structure, they most likely developed through convergent evolution. This notion is further substantiated by a phylogenetic analysis showing that these two polypeptide groups have developed independently (Figure 4). It is also possible that over millions of years of evolution, ABF genes have been exposed to strong selection pressures gaining mutations that assist in combating nematode-specific pathogens. Also, the high incidence of intron gain [25] and different location of introns in nematode genes compared with those of other organisms [32] could account for the different genomic organization between nematode ABFs and invertebrate defensins. Although theoretically possible, common ancestry of nematode ABFs and invertebrate
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MGD-1
L
CSα/β
Intron
ASABF-α
1496 bp
ASABF-β
295 bp
ASABF-γ
295 bp
ASABF-δ
>202 bp
ASABF-ε
CSα/β
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Defensin A
C terminal
>8 bp
ASABF-ζ
>69 bp
ABF-1
712 bp
ABF-2 ABF-3
167 bp
ABF-4
54 bp
ABF-5 ABF-6
54 bp 51 bp
Figure 3. Schematic gene organization of nematode ABFs. The coding region for the leader peptide (L) appears in black. The region encoding the CSa/b and C-terminal regions are grey and hatched, respectively. The intron is depicted as a white bar and its size in base pairs is designated. The relative size of all gene regions, except for that of the intron, is drawn to scale. ASABF-a, accession number AB029816; ASABF-b, accession number AB029812; ASABF-g, accession number AB029813; ASABF-d, accession numbers AB029814, AB029815; ASABF-3, accession number AB090347S1; ASABF-z, accession number AB090349S1; ABF-1 and ABF-2, accession number NC_003279; ABF-3 and ABF-4, accession number NC_003283; ABF-5 and ABF-6, accession number NC_003284. Inset: the cysteine-stabilized a/beta structural motif common to all arthropod and mollusk defensins. The structure of the Mediterranean mussel Mytilus galloprovincialis defensin 1 (MGD-1; left) and the fly Protophormia terraenovae Defensin A (right) (PDB entries 1FJN and 1ICA, respectively) are presented. Alpha helices are presented in green; b strands in blue. The models were generated using the RasMol Version 2.6 software.
defensins would have had to involve a large number of events. These events would have had to include losing the introns flanking the shuffling exon, gaining an intron in the middle of the exon encoding the mature ABFs, and gaining a large number of mutations. Therefore, although still putative, convergent evolution seems a more feasible trajectory for the development of nematode ABFs and invertebrate defensins. All reported CSa/b-containing antibacterial polypeptides are translated as precursors containing a signal peptide at their N terminus to ensure secretion. Some CSa/b-containing antimicrobial peptides also contain a pro-region, which is removed post-translationally to generate mature peptides before secretion. The pro-region is usually located between the signal peptide and the mature region in some CSa/b-containing antimicrobial peptides (insect defensins) [6]. In ABFs, the secretory signal is located N-terminal to the mature region and the pro-region is C-terminal to the mature peptide. This organization, although different than that of insect defensins, is similar to that of MGD-1 and myticin A, www.sciencedirect.com
two mollusk defensins [11,33]. This finding instigated Zhang and Kato [11] to suggest that nematode ABFs and mollusk MGD-1 and myticin A are genetically related. In addition, comparison of ASABF-a, ABF-4, ABF-5 and ABF-6 to other CSa/b-containing polypeptides supported the hypothesis raised by Zhang and Kato [11] because gene organization and the cysteine array of nematode ABFs are similar to that of the mollusk defensin MGD-1 and myticin A [34]. Recently, we suggested that the exon encoding the mature MGD-1 is genetically related to mature arthropod defensins because they share high similarity in sequence, structure, bioactivity and genomic organization [6]. In addition, as described earlier, invertebrate (arthropod and mollusk) defensins and nematode ABFs developed independently throughout evolution (Figure 4). Therefore, it is unlikely that MGD-1 and nematode ABFs are genetically related. Although proposed by Zhang and Kato [11], the authors also add that there is no highly reliable evidence, such as sequence identity or conservative genomic organization, for common ancestry of MGD-1 and ABFs.
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Dm Aa-B Lq Om-A MGD-1 ASABF-α ASABF-β ASABF-γ ASABF-δ ASABF-ζ ABF-2 ABF-1 ASABF-ε ABF-3 ABF-5 ABF-4 ABF-6 Figure 4. Phylogenetic tree of invertebrate defensins and nematode ABFs. The protein regions taken for analysis were the regions encoding the CSa/b. ASABF-a, accession number BAA89497; ASABF-b, accession number BAC00497; ASABF-g, accession number BAC00498; ASABF-d, accession numbers BAC00499; ASABF-3, accession number BAC41495; ASABF-z, accession number BAC57992; ABF-1, accession number AAK68260; ABF-2, accession number AAK68258; ABF-3, accession number NP_506950; ABF-4, accession number NP_507965; ABF-5, accession number NP_510136; ABF-6, accession number NP_741914. Aa-B, Aedes aegypti defensin B (accession number AAB35030); Dm, Drosophila melanogaster (accession number S43228); Lq, Leiurus quinquestriatus (accession number JN0613); MGD-1, Mytilus galloprovincialis defensin 1 (accession number S74088); Om-A, Ornithodoros moubata defensin A (accession number BAB41028).
Concluding remarks Sequence identity, bioactivity, structural commonality and genomic organization revealed that nematode ABFs are genetically related [9]. ABF gene analysis revealed that they might have gained an intron along evolution, before duplication and diversification of the genes. Recently, we have suggested that invertebrate (arthropod and mollusk) defensins are genetically related and have most likely evolved via exon shuffling [6,17]. Although nematode ABFs display similarity in activity and structure to invertebrate defensins, lack of sequence similarity and the different genomic organization suggest that these two polypeptide families exhibit an example of convergent evolution. To have a general picture of the evolutionary trajectory of antibacterial polypeptides, researchers must invest more time to isolate and analyze new members from other phyla. Comparative analysis of the genomic organization of a large number of genes will enable us to determine whether they exhibit common ancestry or convergent evolution. Thus, future isolation and analysis of novel genes encoding antibacterial polypeptides could hold the key to the mysterious phylogenetic trajectory of these unique molecules.
References 1 Schulenburg, H. et al. (2004) Evolution of the innate immune system: the worm perspective. Immunol. Rev. 198, 36–58 2 Kato, Y. (1995) Humoral defense of the nematode Ascaris suum: antibacterial, bacteriolytic and agglutinating activities in the body fluid. Zoolog. Sci. 12, 225–230 3 Kimbrell, D.A. and Beutler, B. (2001) The evolution and genetics of innate immunity. Nat. Rev. Genet. 2, 256–267 4 Hoffmann, J.A. and Reichhart, J.M. (2002) Drosophila innate immunity: an evolutionary perspective. Nat. Immunol. 3, 121–126 www.sciencedirect.com
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5 Mallo, G.V. et al. (2002) Inducible antibacterial defense system in C. elegans. Curr. Biol. 12, 1209–1214 6 Froy, O. and Gurevitz, M. (2003) Arthropod and mollusk defensins – evolution by exon-shuffling. Trends Genet. 19, 684–687 7 Kato, Y. and Komatsu, S. (1996) ASABF, a novel cysteine-rich antibacterial peptide isolated from the nematode Ascaris suum. Purification, primary structure, and molecular cloning of cDNA. J. Biol. Chem. 271, 30493–30498 8 Kato, Y. et al. (2002) abf-1 and abf-2, ASABF-type antimicrobial peptide genes in Caenorhabditis elegans. Biochem. J. 361, 221–230 9 Pillai, A. et al. (2003) Induction of ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides by bacterial injection: novel members of ASABF in the nematode Ascaris suum. Biochem. J. 371, 663–668 10 Andersson, M. et al. (2003) Ascaris nematodes from pig and human make three antibacterial peptides: isolation of cecropin P1 and two ASABF peptides. Cell. Mol. Life Sci. 60, 599–606 11 Zhang, H. and Kato, Y. (2003) Common structural properties specifically found in the CSalphabeta-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin? Dev. Comp. Immunol. 27, 499–503 12 Zhang, H. et al. (2000) In vitro antimicrobial properties of recombinant ASABF, an antimicrobial peptide isolated from the nematode Ascaris suum. Antimicrob. Agents Chemother. 44, 2701–2705 13 Otvos, L., Jr. (2000) Antibacterial peptides isolated from insects. J. Pept. Sci. 6, 497–511 14 Matsuyama, K. and Natori, S. (1990) Mode of action of sapecin, a novel antibacterial protein of Sarcophaga peregrina (flesh fly). J. Biochem. (Tokyo) 108, 128–132 15 Cociancich, S. et al. (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268, 19239–19245 16 Maget-Dana, R. and Ptak, M. (1997) Penetration of the insect defensin A into phospholipid monolayers and formation of defensin A-lipid complexes. Biophys. J. 73, 2527–2533 17 Froy, O. and Gurevitz, M. (1998) Membrane potential modulators: a thread of scarlet from plants to humans. FASEB J. 12, 1793–1796 18 Hanzawa, H. et al. (1990) 1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin. FEBS Lett. 269, 413–420 19 Bonmatin, J.M. et al. (1992) Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding. J. Biomol. NMR 2, 235–256 20 Cornet, B. et al. (1995) Refined three-dimensional solution structure of insect defensin A. Structure 3, 435–448 21 Yang, Y.S. et al. (2000) Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1). Biochemistry 39, 14436–14447 22 Froy, O. and Gurevitz, M. (2003) New insight on scorpion divergence inferred from comparative analysis of toxin structure, pharmacology, and distribution. Toxicon 42, 549–555 23 Froy, O. and Gurevitz, M. (2004) Arthropod defensins illuminate the divergence of scorpion neurotoxins. J. Pept. Sci. 10, 714–718 24 Aizawa, T. et al. (2000) Structural analysis of an antibacterial peptide derived from a nematode. In Peptide Science (Shioiri, T., ed.), pp. 269–272, The Japanese Peptide Society 25 Logsdon, J.M., Jr. et al. (1995) Seven newly discovered intron positions in the triose-phosphate isomerase gene: evidence for the introns-late theory. Proc. Natl. Acad. Sci. U. S. A. 92, 8507–8511 26 Logsdon, J.M., Jr. (2004) Worm genomes hold the smoking guns of intron gain. Proc. Natl. Acad. Sci. U. S. A. 101, 11195–11196 27 Coghlan, A. and Wolfe, K.H. (2004) Origins of recently gained introns in Caenorhabditis. Proc. Natl. Acad. Sci. U. S. A. 101, 11362–11367 28 Qiu, W.G. et al. (2004) The evolutionary gain of spliceosomal introns: sequence and phase preferences. Mol. Biol. Evol. 21, 1252–1263 29 Patthy, L. (1994) Introns and exons. Curr. Opin. Struct. Biol. 4, 383–392 30 Patthy, L. (1999) Genome evolution and the evolution of exon-shuffling – a review. Gene 238, 103–114 31 Ponting, C.P. and Russell, R.R. (2002) The natural history of protein domains. Annu. Rev. Biophys. Biomol. Struct. 31, 45–71
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TRENDS in Microbiology
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Elsevier celebrates two anniversaries with gift to university libraries in the developing world In 1580, the Elzevir family began their printing and bookselling business in the Netherlands, publishing works by scholars such as John Locke, Galileo Galilei and Hugo Grotius. On 4 March 1880, Jacobus George Robbers founded the modern Elsevier company intending, just like the original Elzevir family, to reproduce fine editions of literary classics for the edification of others who shared his passion, other ’Elzevirians’. Robbers co-opted the Elzevir family’s old printer’s mark, visually stamping the new Elsevier products with a classic old symbol of the symbiotic relationship between publisher and scholar. Elsevier has since become a leader in the dissemination of scientific, technical and medical (STM) information, building a reputation for excellence in publishing, new product innovation and commitment to its STM communities. In celebration of the House of Elzevir’s 425th anniversary and the 125th anniversary of the modern Elsevier company, Elsevier will donate books to 10 university libraries in the developing world. Entitled ‘A Book in Your Name’, each of the 6 700 Elsevier employees worldwide has been invited to select one of the chosen libraries to receive a book donated by Elsevier. The core gift collection contains the company’s most important and widely used STM publications including Gray’s Anatomy, Dorland’s Illustrated Medical Dictionary, Essential Medical Physiology, Cecil Essentials of Medicine, Mosby’s Medical, Nursing and Allied Health Dictionary, The Vaccine Book, Fundamentals of Neuroscience, and Myles Textbook for Midwives. The 10 beneficiary libraries are located in Africa, South America and Asia. They include the Library of the Sciences of the University of Sierra Leone; the library of the Muhimbili University College of Health Sciences of the University of Dar es Salaam, Tanzania; the library of the College of Medicine of the University of Malawi; and the libraries of the University of Zambia, Universite du Mali, Universidade Eduardo Mondlane, Mozambique; Makerere University, Uganda; Universidad San Francisco de Quito, Ecuador; Universidad Francisco Marroquin, Guatemala; and the National Centre for Scientific and Technological Information (NACESTI), Vietnam. Through ‘A Book in Your Name’, the 10 libraries will receive approximately 700 books at a retail value of approximately 1 million US dollars.
For more information, visit www.elsevier.com www.sciencedirect.com