Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily.

Insects have an efficient defense system against infections. Their antibacterial immune proteins have been well characterized. However, the molecular mechanisms by which insects recognize foreignness are not yet known. Data are presented showing that hemolin (previously named P4), a bacteria-inducible hemolymph protein of the giant silk moth Hyalophora cecropia, belongs to the immunoglobulin superfamily. Functional analyses indicate that hemolin is one of the first hemolymph components to bind to the bacterial surface, taking part in a protein complex formation that is likely to initiate the immune response.

Gene-encoded peptide antibiotics and innate immunity. Do 'animalcules' have defence budgets?

Gene-encoded peptide antibiotics have been isolated from plants, animals and microbes. Their protective role has been related to innate immunity, which has gradually become accepted across the biomedical community. The evidence for the immune function of peptide antibiotics has been convincingly demonstrated by a combination of both in vitro and in vivo data for plants and insects, but for vertebrates in vivo data are scarce. Using frogs as model systems, it has been shown that the genes for antibacterial peptides are down-regulated by glucocorticoids, while IkappaB alpha is clearly up-regulated. Experimental infections with frog bacteria have shown that the normal capacity to control the natural flora is lost after treatment with glucocorticoids. A low-specificity immune mechanism is cost-effective, something that may have been of importance during animal evolution.

The origin of cecropins; implications from synthetic peptides derived from ribosomal protein L1.

We recently showed that Helicobacter pylori grown on plates produce cecropin-like antibacterial peptides to which H. pylori is resistant. This antibacterial activity was traced to fragments from the N-terminus of ribosomal protein L1 (Pütsep et al., Nature, April 22, 1999). The evolutionary suggestion from this finding has now been extended by the synthesis of eight peptides with sequences taken from the N-terminus of ribosomal protein L1 (RpL1) of five different species. Two peptides of different length derived from H. pylori RpL1 showed a potent antibacterial activity, while a peptide with the sequence from Escherichia coli was 20 times less active. Like cecropins the H. pylori peptides were not cytolytic. We suggest that the cecropins have evolved from ribosomal protein L1 of an ancestral intracellular pathogen that developed to a symbiont ending as an organelle. When the R1 gene moved into the host nucleus, a duplication provided a copy from which today cecropins could have evolved.

Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-kappaB and NF-IL6 binding sites in its promoter.

The sequence of a gene from Bombina orientalis was determined which codes for antibacterial peptides. The gene comprises two exons separated by a large intron. Exon 1 codes for the signal peptide, while exon 2 contains the genetic information for two identical bombinins and one bombinin H. The promoter region of the bombinin gene contains putative recognition sites for nuclear factors, such as NFkappaB and NF-IL6. In vivo experiments on B. orientalis have shown that a short contact with bacteria is sufficient to induce a marked increase in the amount of antibacterial peptides in the skin secretion of frogs. This increase was suppressed by pretreatment with glucocorticoids. In the latter case, a significant increase of I kappaB alpha in the secretion is also detectable.

Effects on electrophoretic mobility and antibacterial spectrum of removal of two residues from synthetic sarcotoxin IA and addition of the same residues to cecropin B.

Cecropin B and cecropin IA (sarcotoxin IA) are 35- and 39-residue antibacterial peptides from a silk moth and a meat fly, respectively. Using solid phase synthesis we have made these peptides as well as two 37-residue analogs, one containing a deletion of leucine and lysine (residues 2a and 2b) as compared to cecropin IA, the other containing an insertion of leucine and lysine at the corresponding place in cecropin B. This addition and removal of a lysine residue did not cause the expected change in electrophoretic mobility. When tested for antibacterial spectra, the insertion analog was found to be as active as the parent compound while the deletion analog had lost most of its antibacterial capacity. In addition it was shown that the C-terminal amide contributes to the broad spectrum properties of the cecropins.

Effect of glucocorticoids on the synthesis of antimicrobial peptides in amphibian skin.

Gene-encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF-kappaB is an important transcription factor for many immunity-related mammalian proteins and also for insect immune genes. The activity of NF-kappaB is regulated by the interaction with an inhibitor, I kappaB. It was recently demonstrated that glucocorticoids induce the synthesis of I kappaB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF-kappaB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of I kappaB alpha. These results suggest that also in vertebrates peptide-mediated innate immunity is controlled by NF-kappaB-regulated transcription.

Shortened cecropin A-melittin hybrids. Significant size reduction retains potent antibiotic activity.

We have earlier reported two 26-residue antibacterial peptides made up from different segments of cecropin A (CA) and melittin (M). We now report a substantial reduction in size at the C-terminal section of the highly active hybrid CA(1-8)M(1-18), leading to a series of 20-, 18- and 15-residue analogs with antibiotic properties similar to the larger molecule. In particular, the 15-residue hybrids CA(1-7)M(2-9), CA(1-7)M(4-11) and CA(1-7)M(5-12) are the shortest cecropin-based peptide antibiotics described so far, with antibacterial activity and spectra similar or better than cecropin A and a 60% reduction in size. Their reduced size and highly alpha-helical structure require an alternative mechanism for their interaction with bacterial membranes.

Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids.

Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(1-13)-melittin(1-13) was 100-fold more active than cecropin A against Staphylococcus aureus. It was also a 10-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.

On the primary structures of lysozyme, cecropins and attacins from Hyalophora cecropia.

Diapausing pupae of Cecropia respond to a bacterial infection by the selective synthesis of RNA and 15-20 hemolymph proteins. Of these we have purified lysozyme and two classes of antibacterial proteins called cecropins and attacins. The primary structure has been determined for the lysozyme, one attacin and five cecropins. We have also prepared a cDNA bank, isolated and sequenced clones corresponding to the lysozyme, the two main attacins and one cecropin. The results of these structural studies are briefly summarized. Finally we review the solid phase synthesis of cecropin A and B and 9 analogs of cecropin A.

PR-39, a proline-rich peptide antibiotic from pig, and FALL-39, a tentative human counterpart.

The peptide antibiotic PR-39 was originally isolated from the upper part of pig intestine. It has antibacterial activity against Gram negative bacteria at concentrations comparable with tetracycline. Studies of the mechanism of action showed that PR-39 inhibits both DNA and protein synthesis. Recently, PR-39 was found in wound fluid and was shown to have inductive activity on matrix components as part of the wound repair process. We have now sequenced the complete gene and possible mediators of its expression will be discussed. Our attempts to characterize the human counterpart of PR-39 by probing for the well conserved prepro-part led to a different peptide antibiotic. A clone containing the coding information for this new peptide was isolated from a human bone marrow cDNA library. The putative human peptide antibiotic was designated FALL-39 after the first four residues and the total number of residues. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family with three disulfide bridges, while FALL-39 lacks cysteine. The clone for the prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the peptide. In the presence of the basal medium E, synthetic FALL-39 was highly active against Escherichia coli D21 and Bacillus megaterium Bm11. Residues 13-34 in FALL-39 can be predicted to form a perfect amphipatic helix and CD spectra showed that medium E induced 30% helix formation in FALL-39. By Northern blot analyses the transcript was located in bone marrow and testis. The structure of the gene and the chromosomal location is under investigation.

NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells.

NK-lysin (NKL), a 78-residue antimicrobial peptide, was isolated from pig small intestine. Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds. The sequence showed 33% identity with a part of a putative gene product (NKG5) from activated T and NK cells, NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-1, a NK sensitive tumour cell line, while sheep red blood cells were unaffected. The cDNA clone corresponding to NK-lysin has been characterized. We have also analyzed the cell and tissue specific expression and the induction of the gene. A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2), showed a 30-fold increase of the NKL transcript. NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon. Immunostaining showed NKL to be present in different types of lymphocytes. Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells.

Innate immunity and the normal microflora.

This paper discusses the following ten subtitles with the contents indicated. 1. To meet a microbe: discusses the four alternatives in host-microbe interactions. 2. Receptors and signal transduction giving gene activation: discusses the lipopolysaccharide receptor and the limitations of cell cultures versus use of live animals. 3. Effector molecules--antimicrobial peptides with and without cysteines. A data base exists with over 500 sequences. This paper gives a general overview of five classes of gene-encoded effector molecules, based on the absence or presence of cysteines. These molecules are peptide antibiotics with wide spectra against different microbes. They are synthesized as propeptides and post-translational modifications are common. 4. Effectors of innate immunity--lethal action without host damage: evaluates current opinions about the mode of action of peptide antibiotics and the fact that these effectors do not create host damage. 5. Genes, introns and movable elements. Two cecropin genes containing movable elements and the human cathelicidin gene for proFALL-39/hCAP18 are discussed. 6. The natural microflora. Hippos or frogs as model systems. This section includes the isolation of bacteria from the normal flora of frogs; Aeromonas hydrophila, the bacterium found on all five frog species studied; arguments and selected examples of frog-microbe interactions in vivo and in vitro; and the use of glucocorticoids as control for nuclear factor-kappa B/I kappa B alpha regulation of effector genes. 7. The use of germ-free mice--hard facts from hard work: summarizes new findings which indicate that germ-free mice are born with a set of antibacterial peptides in their small intestine. The intestine of germ-free mice monoinfected with A. hydrophila have peptide patterns that differ depending on a pretreatment with cortisone. 8. Looking back--an evolutionary perspective on innate immunity: arguments for an early evolutionary need for gene-encoded antibacterial factors. Caenorhabditis elegans should provide some answers. The finding of cecropin-like peptides in Helicobacter pylori and the indications that cecropins are derived from ribosomal protein L1. 9. What about viruses? Arguments for the lack of innate immunity against viruses. 10. Five questions floating in the pond of immunology. The normal microflora, its size and control are too often left out from immunological thinking. Animal model systems may sometimes invite misinterpretation. Which animal species are more equal than others?

Antibacterial peptides: basic facts and emerging concepts.

Antibacterial peptides are the effector molecules of innate immunity. Generally they contain 15-45 amino acid residues and the net charge is positive. The cecropin type of linear peptides without cysteine were found first in insects, whilst the defensin type with three disulphide bridges were found in rabbit granulocytes. Now a database stores more than 800 sequences of antibacterial peptides and proteins from the animal and plant kingdoms. Generally, each species has 15-40 peptides made from genes, which code for only one precursor. The dominating targets are bacterial membranes and the killing reaction must be faster than the growth rate of the bacteria. Some antibacterial peptides are clearly multifunctional and an attempt to predict this property from the hydrophobicity of all amino acid side chains are given. Gene structures and biosynthesis are known both in the fruit fly Drosophila and several mammals. Humans need two classes of defensins and the cathelicidin-derived linear peptide LL-37. Clinical cases show that deficiencies in these peptides give severe symptoms. Examples given are morbus Kostmann and atopic allergy. Several antibacterial peptides are being developed as drugs.