Ajitha Pillai et al. (2005) International Worm Meeting "Evolution of nematode ASABF and cecropin-type antimicrobial peptides"

Ajitha Pillai, Yusuke Kato, Hong Zhang

[International Worm Meeting, 2005]

Many organisms including nematodes produce antimicrobial peptides, which are selectively toxic to microbes, for defense against microbial infection. The antimicrobial peptides are categorized based on chemical structure (alpha-helical, CS alpha beta, etc). Although the antimicrobial peptides whose chemical structure is similar can be found in evolutionarily distant organisms, their phylogenetic relationship is often ambiguous due to higher sequence divergence caused by competitive evolution against pathogens. Previously, we reported the alpha-helical antimicrobial peptide, nematode cecropins, as positively induced factors by bacterial injection in the pig round worm, Ascaris suum. Peptides similar to nematode cecropins have been reported in insects (insect cecropins) and tunicates (styelins). Although insect cecropins and styelins are similar near their secretory signal-mature peptide junction, the C-terminal acidic pro-region is found only in styelins but not in insect cecropins [1]. We determined 9 precursors of nematode cecropins. All nematode cecropin precursors contained the C-terminal acidic pro-regions observed in styelins. In addition, the length of each region (secretory signal, mature peptide, and acidic pro-region) was almost identical between nematode cecropins and styelins. The criteria of sequence similarity, precursor organization, and regional length suggest that nematode, insect, and tunicate cecropin-type antimicrobial peptides could have diversified from a common ancestor, opposite to what we previously expected (Pillai et al., 2004 East Asia Meeting). Moreover, another nematode antimicrobial peptide, ASABF, is specifically similar to mollusk antimicrobial peptides, MGDs and myticins. These results suggest that immunity by these antimicrobial peptides observed in nematodes cannot be adaptive convergence but generated in the early stage of animal evolution and still function in some organisms. [1] Zhao et al. (1997) FEBS Lett. 412, 144.

Ajitha Pillai et al. (2003) International Worm Meeting "Induction of ASABF-type antimicrobial peptides by bacterial injection: Novel members of ASABF in the nematode Ascaris suum."

Ajitha Pillai, Yusuke Kato, Hong Zhang, Satoshi Ueno

[International Worm Meeting, 2003]

Antigen-specific adaptive immunity is found only in higher vertebrates. In contrast, innate immunity against non-specific targets, e.g., production of antimicrobial peptides, is observed in a wide range of animals. Thus, some invertebrate models are thought to be useful for studying innate immunity, e.g., insects and ascidians. Nematodes, including the genetic model Caenorhabditis elegans, seem to be potential candidates because of their experimental utility. To achieve this possibility, it is prerequisite to confirm that nematodes can recognize non-selves, especially microbes, and actively remove them. Recently, some specific responses induced by contact with pathogens were reported for C. elegans. A bacterial pathogen, Microbacterium nematophilum, induces a morphological change in the post-anal region (Hodgkin et al., 2000). Contact with another bacterial pathogen, Serratia marcescens, induced some specific gene expression including lysozyme- and lectin-like genes (Mallo et al., 2002). However, these responses were observed only in contact with pathogens and not with non-pathogenic microbes, i.e., C. elegans is cultured on a lawn of a non-pathogenic bacterium, E. coli OP50, in laboratories, but such responses have not been detected. Thus, it is still ambiguous whether these pathogen-induced responses are typical innate immune responses to non-specific targets or are specific pathologic changes caused by these pathogens. To test whether nematodes can recognize and respond to microbes in the pseudocoelom (body cavity), injection of heat-killed non-pathogenic bacteria can be used as the immunization usually used in insects. Although C. elegans is an excellent genetic model organism, it is difficult to collect a sufficient number of bacteria-injected individuals and quantify tissue-specific gene expression because of its small size. In contrast, A suum is big enough (adult female is 25-35 cm in length) and is suitable for these experiments, so we previously suggested the advantage of combinational analysis using C. elegans and A. suum. To date, 7 members have been identified as ASABF-type antimicrobial peptides in A. suum. The transcripts for ASABF-alpha, beta, gamma, delta, and 6Cys-alpha clearly increased in the body wall and also in the intestine for ASABF-delta and 6Cys-alpha, 4 h after injection of heat-killed bacteria into the pseudocoelom (body cavity), suggesting that these peptides are inducible in the acute phase of immune response. These results also suggest that the nematodes can recognize bacteria in the pseudocoelomic fluid and evoke an active immune response.

Ajitha Pillai et al. (2004) East Asia Worm Meeting "Immune defense of worms: Novel immune-inducible antimicrobial peptides, nematode cecropins, in Ascaris suum."

Satoshi Ueno, Yusuke Kato, Ajitha Pillai, Hong Zhang

[East Asia Worm Meeting, 2004]

Nematodes, including Caenorhabditis elegans, produce antimicrobial peptides selectively toxic against microbes. These antimicrobial peptides should defend worms from microbial infection. Previously, we reported that some mRNAs encoding ASABF-type antimicrobial peptides increased in the pig roundworm, Ascaris suum, by injection of heat-killed Escherichia coli OP50. To explore other bacteria-inducible genes, cDNA subtraction was performed for intestinal transcripts. Some positively and negatively induced transcripts were detected. Two distinct antimicrobial peptides were identified as the positively induced molecules. One was ASABF-beta, an ASABF-type antimicrobial peptide which we already reported as bacteria-inducible. Another was a similar molecule to cecropin P1, originally reported as an antimicrobial peptide produced in the porcine intestine but recently corrected as an Ascaris peptide (Andersson et al., 2003). Four cecropin P1-like molecules were identified in A. suum by cDNA cloning based on a parasite EST database search (cecropin P1, P2, P3, and P4. Cecropin P4 was detected in the cDNA subtraction). All "nematode cecropins" were induced in the intestine by bacterial injection. In addition, these transcripts were also detected in the body wall, uterus, and ovary. Chemically synthesized nematode cecropins were active against Gram+ and Gram- bacteria, particularly E. coli JM109 and Staphylococcus aureus IFO12732. Moreover, weak activity was observed against the yeast, Saccharomyces cerevisiae. Cecropin-type antimicrobial peptides were first found in insects. Interestingly, precursor organization of nematode cecropins was "secretory signal-mature region-proregion", distinct from those of insect cecropins, suggesting that nematode cecropins are not homologs of insect cecropins.

Satoshi Ueno et al. (2002) Japanese Worm Meeting "Immune defense of worms: ASABF-epsilon and zeta, and ASABF-6Cys-alpha, novel members of ASABF-type antimicrobial peptides in Ascaris suum"

Ajitha Pillai, Yusuke Kato, Satoshi Ueno, Takao Igarashi

[Japanese Worm Meeting, 2002]

Satoshi Ueno et al. (2004) East Asia Worm Meeting "Immune defense of worms: Another novel immune-inducible antimicrobial peptide, ASABF-6Cys-alpha, in Ascaris suum."

Ajitha Pillai, Masaomi Minaba, Yusuke Kato, Hong Zhang, Satoshi Ueno

[East Asia Worm Meeting, 2004]

ASABFs are antimicrobial peptides isolated from nematodes including Caenorhabditis elegans. ASABF-type antimicrobial peptides contain an alpha-helix and a pair of anti-parallel beta-sheets stabilized by 4 intramolecular disulfide bridges. Previously, we reported that mRNAs encoding ASABF-alpha, beta, gamma, and delta increased in the pig roundworm, Ascaris suum, by injection of heat-killed Escherichia coli OP50. On the other hand, we have also reported ASABF-6Cys-alpha containing only 3 disulfide bridges. We tested the immune-inducibility of ASABF-6Cys-alpha. The transcripts encoding ASABF-6Cys-alpha was dramatically induced in the body wall, intestine, and reproductive organs. Especially, over 100 times increment was observed in the body wall and intestine. We are presently trying to prepare a recombinant ASABF-6Cys-alpha and characterize its biological activities.