An Arthropod Defensin Expressed by the Hemocytes of the American Dog Tick, Dermacentor variabilis (Acari: Ixodidae)

Both soluble and cell-mediated components are involved in the innate immune response of arthropods. Injection of Borrelia burgdorferi, the Lyme disease agent, results in the secretion of defensin into the hemolymph of the ixodid tick, Dermacentor variabilis. The presence of the peptide is observed as early as 15 min post-challenge and remains present through 18 h post-challenge. As observed in insects and soft ticks, the transcript for defensin is detected as early as 1 h post-challenge in D. variabilis. RT-PCR resulted in an amplicon of 624 bp with a 225 bp region that translates to a 74 amino acid preprodefensin. The defensin encoding region was amplified, cloned and sequenced from the hemocytes. It appears as though defensin is stored in the granulocytes of the hemolymph and secreted into the hemolymph upon bacterial insult. The role of defensin as a contributing factor in determining vector competency is discussed

Using RNA Interference to Determine the Role of Varisin in the Innate Immune System of the Hard Tick Dermacentor variabilis (Acari: Ixodidae).

Defensins are an important component of the innate immune system of ticks. These small peptides are produced by various genera of ticks, and expressed in various tissues. In this study we used RNA interference to silence the expression of the defensin varisin produced by the hemocytes of the American dog tick, Dermacentor variabilis. Ticks were injected with double stranded varisin RNA prior to being placed on a rabbit. After feeding, the ticks were removed, bled, and the hemolymph plasma and hemocytes separated. Hemocytes were screened for the presence (or absence) of both varisin transcript and peptide. Varisin peptide was below detectable levels and the transcript showed a greater than 99% knockdown. The antimicrobial activity of the hemolymph plasma was reduced 2-4 fold compared to that of control injected ticks indicating varisin accounts for a large portion of the antimicrobial activity of the hemolymph

In silico screening, analysis, and modelling for a novel anticancer peptide

Cancer is currently one of the leading causes of mortality and morbidity worldwide. Most anticancer therapies rely on small molecule drugs (\u3c0.5 kDa). As with all small molecule drugs, chemotherapy is highly toxic and presents many off-target side effects. Peptide drugs offer improved specificity and are cheaper and more accessible to manufacture. In this study, we have developed a support vector machine (SVM) model in order to detect peptide sequences with potential anticancer activity through scanning the Red Sea Metagenomic library. Furthermore, we conducted an in silico study in order to analyze one of the peptides returned by the SVM pipeline and assessed its cytotoxicity and the mode of cell death by conducting MTT and Annexin V staining assays, respectively. We observed that the selected anticancer peptide contains the C-terminal portion of the homeodomain structure, of human Pax6, an antennapedia homeodomain region, and can bind DNA. Furthermore, we observed dose-response cytotoxicity of HepG2 cells with our peptide. No such cytotoxicity was observed in HeLa cells; a morphological change, however, was observed. We examined the cytotoxicity of our drug against 1BR-hTERT normal skin cells. Our peptide drug induced dose-dependent cytotoxicity that was markedly weaker than that of cancer treated cells. Together our data illustrates the isolation of one peptide drug candidate from the AUC Red Sea metagenomic library; furthermore, we were able to observe the selective dose-dependent reduction of HepG2 cell viabilit

Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus

<p>Abstract</p> <p>Background</p> <p>The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule.</p> <p>Results</p> <p>Two isoforms of the defensin gene <it>(def1 </it>and <it>def2</it>) were identified in the European tick <it>Ixodes ricinus</it>. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values) against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established.</p> <p>Conclusions</p> <p>Although there is nothing known about the realistic concentration of defensins in <it>I. ricinus </it>tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.</p

A Defensin-Like Gene Expressed in the Black-Legged Tick, Ixodes scapularis (Acari: Ixodidae)

The black-legged tick Ixodes scapularis linnaeus (Acari: Ixodidae) is an important vector of microbial pathogens. Knowledge of the tick\u27s innate immune response, particularly defensin and other antimicrobial peptides, is important for understanding how microbes survive in this tick. A defensin gene (slnA) from I. scapularis was obtained by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA extracted from tissues of female ticks. RT-PCR indicated the gene was expressed in the midgut, haemocytes, and fat-body, although no evidence of a peptide was found. Sequencing a cloned cDNA fragment revealed a 225 bp open reading frame encoding a 74 amino acid pre-prodefensin, including the putative 38 amino acid mature peptide. Similarity between the defensin amino acid sequences of I. scapularis and Dermacentor variabilis (Say) (Acari: Ixodidae) was 62.2% for the pre-prodefensin region; for the mature defensins from these two species the similarity was 78.9%, with the six cysteine residues being located in the same relative position. PCR amplification and sequencing of chromosomal DNA suggests that slnA, along with vsnA, the defensin gene from D. variabilis, does not contain any introns. This is in contrast to the defensins described for the soft tick, Ornithodoros moubata (sensu Walton) (Acari: Argasidae). The role of defensin in the innate immune response of I. scapularis following microbial invasions is discussed

Molluscan antimicrobial peptides, a review from activity-based evidences to computer-assisted sequences

Antimicrobial peptides (AMPs) represent the most universal immune effectors. Molluscs constitute the second largest animal phylum, after Arthropods, in term of number of species. Only a negligible number has been investigated regarding AMPs. The choice of the species to be studied relied on their economical importance and availability. First studies on molluscan AMPs dated from 1996 and were based on biological activities of biochemical-purified fractions. Such approach released all the original structures we know, with biological activity sometimes different from one isoform to another. Then, molecular biology techniques were applied to molluscan AMPs starting in 1999. Complete screening of genome expression in various situations became available, as well as some exotic Molluscs, the ones collected from deep-sea hydrothermal vent, for instance. Full sequences of active peptides and precursors, and gene organizations were established. A breakthrough consisted in the discovery of numerous AMP variants, even within the same animal. In addition, computer homology revealed the existence of already known AMPs in new studied molluscan species

Purification and characterization of a novel salivary antimicrobial peptide from the tick, \u3cem\u3eIxodes scapularis\u3c/em\u3e

A novel antimicrobial peptide was isolated from the saliva of the Lyme disease tick vector, Ixodes scapularis, henceforth designated as ISAMP (I. scapularis Antimicrobial Peptide). ISAMP was purified using a sequential method including ultra filtration, gel filtration and reverse-phase high performance liquid chromatography. The purified peak had a molecular weight of 5.3 kDa by MALDI/TOF-MS and its amino acid sequence, determined by Edman degradation was PDxGxPxxVKAGRxPxxSI. A BLASTP search revealed that the protein is a putative 5.3 kDa secreted protein (AAM93656) from I. scapularis. The predicted protein is composed of 69 amino acids with no conserved domain motifs. Purified ISAMP was found to have antimicrobial activities against bacteria. Gene expression studies were carried out to observe ISAMP expression in different tick tissues. RT-PCR results indicated that the gene was expressed in hemocytes, fat body and salivary gland but virtually no expression was observed in the midgut. ISAMP is only similar to other Ixodid tick proteins, thus it is a member of a unique family

Tissue and Life Stage Distribution of a Defensin-Like Gene in the Lone Star Tick Amblyomma americanum

The transcript sequence of the Amblyomma americanum Linnaeus (Acari: Ixodidae) defensin, termed amercin (amn), was ascertained and a 219-bp amn coding region identified. The gene encodes a 72-amino acid prepropeptide with a putative 37-amino acid mature peptide. This gene shows little similarity to either of the defensins from Amblyomma hebraeum Koch, the only other Amblyomma species for which a defensin has been described. Sequence comparisons with other tick defensins reveal amn to be shorter (6 bp or 2 amino acids) than the Ixodes scapularis Linnaeus (Acari: Ixodidae) and Dermacentor variabilis (Say) (Acari: Ixodidae) defensin sequences. The amercin prepropeptide has 60.8% and 59.5% similarity with the I. scapularis and D. variabilis prepropeptides, respectively, whereas the mature amercin peptide has 73.7% and 71.1% similarity with the mature peptides of these ticks. Similarity with other tick defensins ranges from 42% to 71%. In A. americanum, defensin transcript was found in the midgut, fat body and salivary gland tissues, as well as in the haemocytes. Defensin transcript was also present in early-stage eggs (less than 48 h old), late-stage eggs (approximately 2 weeks old), larvae and nymphs of A. americanum and I. scapularis, both of which are vector-competent for Borrelia spirochetes

The evolution of immune genes in tsetse flies (Glossina) and insights into tsetse-symbiont-trypanosome interactions

Tsetse flies (genera Glossina) are the sole biological vectors of African Trypanosoma species, the infectious agents of African Trypanosomiasis. Vector control is a key inhibitor of disease transmission; however, long-term control measures are economically and ecologically unsustainable and therefore, alternatives must be explored. In this thesis we aim to explore the evolution of three important immune genes: attacin-A (AttA), Defensin (Def) and Toll-like receptor 2 (TLR2), in relation to symbionts and parasitic interactions. This could in turn lay the foundations for genetic control methods The successful identification of novel attacin orthologues confirmed the previous descriptions of attacin clusters within the Glossina genome, while a single novel defensin orthologue was identified in each of the six Glossina genomes. A total of six TLRs were confirmed within the Glossina genome, and three additional TLRs were potentially identified, though these are unconfirmed. The evolutionary history of the attacin cluster remains undetermined, however concerted evolution likely impacts the evolution of AttA, while Def and TLRs are governed by strict Darwinian selection. A wild population sample of Glossina morsitans morsitans illustrated differing levels of nucleotide variation in each gene, Def being the least polymorphic (n = 8) and TLR2 being the most (n = 22). All genes indicated a recent population expansion event and deviations from neutrality, indicative of population expansion and balancing selection. Genetic variation in both AttA and TLR2 was found to be maintained via purifying selection, while Def exhibited signs of the Red Queen arms race and balancing section. Trypanosome infection rates were unexpectedly high (69.35%), consisting of mixed species infections. Advantageous Def variants were observed to reduce infection rates within samples, while an observable relationship between TLR2 and symbiont variation, and infection rate requires further research. The results within described the impacts of evolution and population change on immune genes and how the interactions with symbiont populations can influence trypanosome infection rates. This thesis indicates that an understanding of the evolution and interactions of the tsetse-symbiont-trypanosome triplet could be used to inform novel genetic control methods

Silencing Expression of the Defensin, Varisin, in Male Dermacentor variabilis by RNA Interference Results in Reduced Anaplasma Marginale Infections

Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections