Antibacterial peptide defensin is involved in midgut immunity of the soft tick,Ornithodoros moubata

Two defensin genes A and B were previously demonstrated to be up-regulated by blood feeding in the soft tick, Ornithodoros moubata [Nakajima et al. (2001) Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Biochem Mol Biol 31: 747-751]. In this study, two defensin isoforms C and D similar to defensins A and B were newly cloned. A total of four defensins have been identified in O. moubata. All four Ornithodoros defensins are coded as prepro-defensins. Ornithodoros defensin genes consist of four exons and three introns, an organization reported in mussel defensins but not insect defensins. Ornithodoros defensin C and D genes are predominantly expressed in the midgut and up-regulated in response to blood feeding. The mature peptide of the previously cloned Ornithodoros defensin A was purified from the midgut lumen, indicating defensin is secreted into the midgut. These findings confirm the involvement of Ornithodoros defensin in midgut immunity.     

Identification and molecular characterization of defensin gene from the ant Formica aquilonia

The effectors of the insect immune system are antimicrobial peptides. With the aim of studying the evolution of immune system genes, we identified a gene encoding the antimicrobial peptide defensin from a social insect, the wood ant Formica aquilonia. In this article we report the identification and characterization of this gene. We also compare the ant defensin gene structure to that previously obtained from two other hymenopteran species, the honeybee, Apis mellifera, and the bumblebee, Bombus ignitus. The ant defensin gene structure differs from both of these bee defensins with respect to the number and length of introns and exons.     

The malaria vector mosquito Anopheles gambiae expresses a suite of larval-specific defensin genes

cDNAs of Anopheles gambiae Defensin 2 ( AgDef2 ), Defensin 3 ( AgDef3 ) and Defensin 4 ( AgDef4 ), identified in the genome sequence, have been characterized and their expression profiles investigated. In contrast to both typical defensins and insect antimicrobial peptides generally, the newly identified defensins were not upregulated with acute-phase kinetics following immune challenge in insects or cell culture. However, mRNA abundance of AgDef2, AgDef3 and AgDef4 increased significantly during the larval stages. Promoter analysis of all three genes failed to identify putative immune response elements previously identified in other mosquito defensin genes. As previous studies failed to identify these larval-specific defensins, it seems likely that further antimicrobial peptide genes with nontypical expression profiles will be identified as more genome sequences become available.     

Antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines

The antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines has been investigated. A peptide corresponding to the HNP-1 sequence without the six cysteines (HNP-1deltaC) exhibited antibacterial activity toward gram-negative and gram-positive bacteria. Truncated analogs wherein the nine N-terminal residues of HNP-1 and the remaining three cysteines were deleted (HNP-1deltaC18) or the G was replaced with A (HNP-1deltaC18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1deltaC and HNP-1deltaC18 in the presence of 100 mM NaCl, except in the case of Pseudomonas aeruginosa. The linear peptides were active in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that proton motive force was not essential for killing of bacteria by the peptides. In fact, in the presence of CCCP, the peptides were active against P. aeruginosa even in the presence of 100 mM NaCl. The antibacterial activity of HNP-1deltaC, but not that of the shorter, 18-residue peptides, was attenuated in the presence of serum. The generation of defensins without cysteines would be easier than that of disulfide-linked defensins. Hence, linear defensins could have potential as therapeutic agents.     

Intraspecific nucleotide variation at the pheromone binding protein locus in the turnip moth, Agrotis segetum

Inter- and intraspecific amino acid variability in the pheromone binding proteins (PBPs) of the Lepidoptera is believed to contribute to a molecular mechanism of pheromone blend discrimination. Messenger RNA coding for PBP sequence in Agrotis segetum (Noctuidae) was cloned, and nucleotide and inferred amino acid variation across a 769-bp region of a PBP locus was studied in two populations. A single gene copy was fully sequenced, revealing an intron/exon structure conserved with distant saturniids. While several nucleotide substitutions are predicted to result in amino acid replacement, tests for the presence of natural selection suggest that the observed variation is neutral. A phylogenetic analysis provides evidence that the two populations are in the process of genetic isolation.     

Immunity proteins from mosquito cell lines include three defensin A isoforms from Aedesaegypti and a defensin D from Aedesalbopictus

An Aedes aegypti mosquito cell line, Aag-2, exhibits a response to immune stimulation that is qualitatively similar to that of C7–10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat-killed, Gram-positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A differential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we defined as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the final amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat-killed bacteria, and defensin mRNA abundance was maximal at 12–36 h after induction.     

Purification, cDNA cloning and expression of an insect defensin from the great wax moth, Galleria mellonella

An insect defensin, named Galleria defensin, was purified from the larval haemolymph of Galleria mellonella immunized against E. coli. The peptide was composed of forty-three amino acid residues containing six cysteines that might be engaged in intramolecular disulphide bridges. The primary structure of Galleria defensin shared about 90.7% identity to that of heliomicin, which was an insect defensin isolated from Heliothis virescens. The full-length cDNA encoding Galleria defensin was cloned from the fat body of the immunized G. mellonella larvae. Northern blot analysis revealed that Galleria defensin was expressed not only in the fat body but also in the midgut against invading bacteria into haemocoel. This is the first report presenting cDNA and expression of an insect defensin in the lepidopteran species.     

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.     

Host-microbe interaction: mechanisms of defensin deficiency in Crohn's disease

Defensins are endogenous antibiotics with microbicidal activity against Gram-negative and -positive bacteria, fungi, enveloped viruses and protozoa. A disturbed antimicrobial defense, as provided by Paneth and other epithelial cell defensins, seems to be a critical factor in the pathogenesis of Crohn's disease, an inflammatory disease of the intestinal tract. Different direct and indirect mechanisms leading to a breakdown of antimicrobial barrier function include direct changes in defensin gene numbers (e.g., copy number polymorphism), genetic mutations in pattern-recognition receptors (e.g., nucleotide-binding oligomerization domain 2) and, as described recently, a differentiation problem of epithelial stem cells mediated by the wingless type (Wnt) pathway. Knowledge regarding the regulation and biology of defensins provides an attractive target to open up new therapeutic avenues.     

Ticks (Ixodidae) from passerine birds in the Carpathian region

Birds have been found to be a reservoir host of borrelia. In order to assess the situation in Slovakia ticks were collected from a total of 3057 mist-netted, ringed and released passerine birds in two locations at 500 m (in 2001) and 1000 m (in 2003) above sea level in the Bukovské Vrchy Hills, part of the Carpathian region in the north-east of Slovakia. A total of 75 birds of 16 species were infested with subadult ticks of Ixodes ricinus species (prevalence of parasitization 5%). Sixty-two larvae from 31 birds of 9 species and 80 nymphs from 52 birds of 15 species were found. The highest intensity of parasitization was observed on blackbirds Turdus merula, song thrushes T. philomelos and dunnocks Prunella modularis. Six Ixodes ricinus adult ticks were found on humans working with birds, and one I. ricinus female tick on their dog. In ticks, the presence of Rickettsia sp., Coxiella burnetii, Borrelia burgdorferi sensu lato and members of the Anaplasmataceae and Piroplasmidae, were investigated by polymerase chain reaction, followed by sequence analysis. Rickettsia sp. was found in 1 nymph from the European robin Erithacus rubecula, in 3 adult ticks (1 male, 2 females) from humans and in the tick from the dog. The closely related Ehrlichia- like species "Schotti variant" was detected in 1 nymph from the song thrush. Borrelia afzelii was identified in 1 male and B. garinii in 1 female tick collected on humans. Ixodes ricinus was found to be the vector of a wide spectrum of tick-borne pathogens in a mountainous area of the Carpathians. Because of the low yield of ticks and pathogens the importance of birds as reservoir hosts is still poorly understood.     

Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations.

We tested the in vitro susceptibility of Candida albicans to three defensins from human neutrophilic granulocytes (HNP-1, 2, and 3), a homologous defensin from rabbit leukocytes (NP-1), and four unrelated cationic peptides. Although the primary amino acid sequences of HNP-1, 2, and 3 are identical except for a single amino-terminal amino acid alteration, HNP-1 and HNP-2 killed C. albicans but HNP-3 did not. C. albicans blastoconidia were protected from HNP-1 when incubations were performed in the absence of oxygen or in the presence of inhibitors that blocked both of its mitochondrial respiratory pathways. Neither anaerobiosis nor mitochondrial inhibitors substantially protected C. albicans exposed to NP-1, poly-L-arginine, poly-L-lysine, or mellitin. Human neutrophilic granulocyte defensin-mediated candidacidal activity was inhibited by both Mg2+ and Ca2+, and was unaffected by Fe2+. In contrast, Fe2+ inhibited the candidacidal activity of NP-1 and all of the model cationic peptides, whereas Mg2+ inhibited none of them. These data demonstrate that susceptibility of C. albicans to human defensins depends both on the ionic environment and on the metabolic state of the target cell. The latter finding suggests that leukocyte-mediated microbicidal mechanisms may manifest oxygen dependence for reasons unrelated to the production of reactive oxygen intermediates by the leukocyte.     

Intramolecular inhibition of human defensin HNP-1 by its propiece.

We examined mechanisms that protect host defense cells from their cytotoxic effector molecules. Human neutrophil peptides (HNP) 1-3 are microbicidal and cytotoxic defensins, initially synthesized as 94-amino acid preproHNP(1-94), cotranslationally proteolyzed to proHNP(20-94), then converted by removal of the anionic propiece to mature HNP(65-94)(HNP-1 and -3) and HNP(66-94) (HNP-2). We hypothesized that during synthesis and subcellular sorting the anionic propiece inhibits the cytotoxicity of the cationic defensin. We expressed preproHNP-1 cDNA in recombinant baculovirus-infected insect cells that secreted the normally transient proHNP-1(20-94) into the medium. Cyanogen bromide cleaved proHNP-1(20-94) at the fortuitously located Met64 to yield mature recombinant HNP-1(65-94) and unlinked propiece. Recombinant and native HNP-1 purified from PMN were identical as judged by mass spectrometry, retention time in reverse-phase high performance liquid chromatography, migration on acid-urea polyacrylamide gels, and reaction with a conformation-specific antibody. Recombinant and native HNP-1 had comparable microbicidal activity towards Listeria monocytogenes and were similarly potent in permeabilizing K562 leukemia cells, but proHNP-1(20-94) was virtually inactive in both assays. Addition of unlinked propiece (proHNP-1(20-64) with Met64-->homoserine) inhibited the bactericidal and cell-permeabilizing activity of mature HNP-1 in a dose-dependent manner. Linked, and to a lesser extent unlinked, propiece interfered with the binding of HNP-1 to target cells. The propiece thus acts as an efficient intramolecular inhibitor of defensin HNP-1 cytotoxicity.     

The white gene of the tephritid fruit fly Bactrocera tryoni is characterized by a long untranslated 5' leader and a 12 kb first intron

A 300 bp fragment from exon 6 of the white gene of Bactrocera tryoni was used to screen a B. tryoni genomic library. One positive (∼14 kb) insert contained exons 2–6 of white by nucleotide and amino acid sequence similarity to the white genes of D. melanogaster (O'Hare et al ., 1984; Pepling & Mount, 1990). Lucilia cuprina (Garcia et al ., 1996). Ceratitis capitata (Zwiebel et al ., 1995) and Anopheles gambiae (Besansky et al ., 1995). A white 5' cDNA fragment containing exons 1, 2 and part of exon 3 was amplified, cloned and sequenced. An inverse PCR fragment of genomic DNA was generated, containing the exon 1 coding region plus ∼2.1 kb of upstream sequence, encompassing the putative promoter of the gene. Exon 1 was found to be 728 bp long, encoding the first twenty-five amino acids. The full length of intron 1 was shown to be 12 kb (amplified using long PCR protocols), up to 3 times the length of the longest white intron 1 isolated to date.     

Comparison of self-assessment and scutal index for the duration of Ixodes ricinus tick attachment

Experimental studies have shown that the efficacy of transmission of B. burgdorferi sensu lato from an infected tick to an experimental animal is significantly associated with the duration of tick attachment. Scutal index is the ratio between body length and scutum width and was found to be an objective indicator of the duration of Ixodes scapularis tick attachment, but no data for I. ricinus have been published. In this preliminary report on 30 volunteers with an attached I. ricinus tick (21 had an adult female tick and 9 a nymph), none developed any sign of Lyme borreliosis but we were able to demonstrate two asymptomatic seroconversions in a six-week follow-up period. Twenty-six (87%) volunteers claimed that they were able to determine the approximate duration of tick attachment; participants with attached adult female ticks estimated the duration of attachment to be 30 (2-90) hours, while those with attached nymphs reported 48 (18-90) hours (p = 0.681). Scutal indices were 3.2 (2-5) for nymphs and 3.2 (1-5) for adult ticks, indicating 60- and 48-hour attachment, respectively. According to the volunteers' assessment and scutal index findings, approximately 80% and 45% of adult female ticks were removed within 48 hours after the tick bite (p = 0.032), respectively, while the corresponding values for nymphs were 85% and 17% (p = 0.053). Our findings were limited by low numbers of volunteers and by the lack of experimental data on the value of the scutal index for estimation of the duration of I. ricinus tick attachment.