Defensin from the ornate sheep tick Dermacentor marginatus and its effect on Lyme borreliosis spirochetes

Expression of the previously reported defensin of the tick Dermacentor marginatus (defDM) was analysed in different organs by RT-PCR. mRNA of the defDM gene was detected in the hemolymph, midgut and salivary glands. Moreover defDM was isolated from the tick hemolymph using RP-HPLC and its sequence was determined by mass spectrometry and Edman degradation. Synthetic peptide was used for determining biological activities. The results showed an anti-Gram-positive bacterial role for the defensin. As D. marginatus ticks appear not to be vectors of the Lyme disease agent of the complex Borrelia burgdorferi sensu lato, we tested the influence of defDM on Borrelia afzelii. There is a very clear borrelicidal activity of the defensin, which is concentration dependent and suggests a possible role in the clearing of Borrelia ingested by D. marginatus ticks.     

Application of representational difference analysis to identify sequence tags expressed by Metarhizium anisopliae during the infection process of the tick Boophilus microplus cuticle

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of plagues, including insects and acari. To identify genes involved in the infection process, representational difference analysis was performed using cDNA generated from germinated conidia of M. anisopliae in the tick Boophilus microplus cuticle, and cDNA generated during fungal growth in glucose-rich medium. Sequence determination of approximately 135 clones and comparison analysis using public databases led to the identification of 34 sequences and 14 expressed sequence tags with known orthologs. As expected, almost all identified sequences showed significant similarity to other fungal genes. The diversity of gene clusters found reflects the participation of several proteins in the early infection process of M. anisopliae in the cattle tick B. microplus.     

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

Summary Wa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.The sequence reported in this paper has been submitted to the EMBL/GenBank through DDBJ (accession no, X52525).     

微小牛蜱凝血酶抑制剂的分离纯化与活性研究

从半饱吸血的微小牛蜱(Boophilusmicroplus)唾液腺中分离纯化到了一种凝血酶抑制剂,用飞行质谱测定该抑制剂的分子量为6752Da,不同于已知的从蜱类来源的丝氨酸蛋白酶抑制剂。这是世界上首次报道的从微小牛蜱唾液腺分离出来的凝血酶抑制剂。该抑制剂对凝血酶表现强烈的抑制活性,对激活的第X因子和胰蛋白酶具有微弱的抑制活性。     

Sequencing a new target genome: the Boophilus microplus (Acari: Ixodidae) genome project

The southern cattle tick, Boophilus microplus (Canestrini), causes annual economic losses in the hundreds of millions of dollars to cattle producers throughout the world, and ranks as the most economically important tick from a global perspective. Control failures attributable to the development of pesticide resistance have become commonplace, and novel control technologies are needed. The availability of the genome sequence will facilitate the development of these new technologies, and we are proposing sequencing to a 4-6X draft coverage. Many existing biological resources are available to facilitate a genome sequencing project, including several inbred laboratory tick strains, a database of approximately 45,000 expressed sequence tags compiled into a B. microplus Gene Index, a bacterial artificial chromosome (BAC) library, an established B. microplus cell line, and genomic DNA suitable for library synthesis. Collaborative projects are underway to map BACs and cDNAs to specific chromosomes and to sequence selected BAC clones. When completed, the genome sequences from the cow, B. microplus, and the B. microplus-borne pathogens Babesia bovis and Anaplasma marginale will enhance studies of host-vector-pathogen systems. Genes involved in the regeneration of amputated tick limbs and transitions through developmental stages are largely unknown. Studies of these and other interesting biological questions will be advanced by tick genome sequence data. Comparative genomics offers the prospect of new insight into many, perhaps all, aspects of the biology of ticks and the pathogens they transmit to farm animals and people. The B. microplus genome sequence will fill a major gap in comparative genomics: a sequence from the Metastriata lineage of ticks. The purpose of the article is to synergize interest in and provide rationales for sequencing the genome of B. microplus and for publicizing currently available genomic resources for this tick.     

Cloning and sequencing of a putative acetylcholinesterase cDNA from Boophilus microplus (Acari: Ixodidae)

Using a strategy based on degenerate primers derived from acetylcholinesterase (AChE) from other species, we cloned and sequenced a putative AChE cDNA from the southern cattle tick, Boophilus microplus (Canestrini). The sequence has a high degree of homology to sequences of AChE from other species reported in the GenBank. The open reading frame of 1,689 bp, corresponding to a deduced sequence of 563 amino acids, has conserved regions and features shared by the AChE family, necessary for its catalytic activity. No differences were found in the putative cDNA sequences from organophosphorus acaricide (OP) resistant and susceptible strains. The results suggest that this putative AChE gene is not involved in resistance to OP compounds as a mutated gene in the resistant strain studied. However, differences were detected, with a probe derived from this cDNA, in DNA fragments after digestion of genomic DNA from different strains with restriction nucleases. This indicates polymorphism in this gene in B. microplus.     

Differential expression of Ixodes ricinus tick genes induced by blood feeding or Borrelia burgdorferi infection

Ixodes ricinus L. is the principal European vector of Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. Subtractive hybridization was used to isolate tick genes that were induced in whole ticks after blood meals on uninfected and B. burgdorferi-infected guinea pigs. Novel cDNA clones with similarity to cytochrome c oxidase, salivary secreted protein, actin, and a cysteine protease propeptide were induced after a blood meal. Novel cDNA clones with similarity to thioredoxin peroxidases, dolichyl-phosphate beta-glucosyltransferase, glutathione S-transferase, defensin, ML domain-containing protein, and von Willebrand factor were induced after B. burgdorferi infection. Virtual Northern analysis was used to verify that these genes were differentially expressed in ticks after a pathogen-infected blood meal and to detect their tissues of expression. The characterization of genes that are induced after an infected blood meal is essential for gaining an understanding of the molecular mechanisms that underlie vector-pathogen interactions.     

Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella

Two hemolymph proteins were isolated from the wax moth, Galleria mellonella, larvae by a two-step procedure consisting of acid extraction and reversed phase (RP)-HPLC. One was an apolipophorin III (apoLp-III) previously characterized as a lipopolysaccharide (LPS) binding protein in the hemolymph of G. mellonella. The other was confirmed to be a new protein with a molecular mass of 23,768.69 Da, referred to as Gm protein-24. The full-length cDNA of Gm protein-24 was cloned from the fat body. The cDNA structure showed that it is a 219-residues protein derived from the precursor of 236 amino acids. The effects of apoLp-III and Gm protein-24 have been tested on the insect humoral immunity. ApoLp-III enhanced the activity of antibacterial peptide such as cecropin but Gm protein-24 had no effect on cecropin activity. On the other hand, Gm protein-24 and apoLp-III were both involved in the activation of prophenoloxidase (PPO) cascade, which has been regarded as a critical immune reaction in insect hemolymph. Of note, the Gm protein-24 was a significantly stronger activator of PPO cascade than apoLp-III.     

Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. The cysteine-rich AMPs such as defensin and hepcidin have been extensively studied from various organisms, but their role in disease defense in catfish is unknown. As a first step, we sequenced a hepcidin cDNA from both channel catfish and blue catfish, and characterized the channel catfish hepcidin gene. The channel catfish hepcidin gene consists of two introns and three exons that encode a peptide of 96 amino acids. The amino acid sequences and gene organization were conserved between catfish and other organisms. In contrast to its almost exclusive expression in the liver in humans, the channel catfish hepcidin gene was expressed in a wide range of tissues except brain. Its expression was detected early during embryonic and larval development, and induced after bacterial infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC) in a tissue-specific manner. The upregulation was observed in the spleen and head kidney, but not in the liver. The expression of hepcidin was upregulated 1--3 days after challenge, but returned to normal levels at 7 days after challenge. The expression profile of the catfish hepcidin gene during the course of bacterial infection mirrors those of inflammatory proteins such as chemokines, suggesting an important role for hepcidin during inflammatory responses.     

Plaque Formation in Tissue Cultures by Rickettsia rickettsi Isolated Directly from Whole Blood and Tick Hemolymph

A simple technique is described for isolation of Rickettsia rickettsi directly from tick hemolymph and whole blood of rickettsemic guinea pigs by means of the plaque assay technique in primary chicken embryo tissue cultures. Plaque-forming units per drop of hemolymph were almost 100-fold greater for partially engorged ticks than for unengorged ticks. Rickettsemia in guinea pigs fed upon by infected ticks was detected as early as 24 hr before fever. No morphological differences were noted between plaques formed by rickettsiae isolated from tick hemolymph or from whole guinea pig blood.     

Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major

Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.