Molecular characterization of a defensin gene from a hard tick, Dermacentor silvarum
© Wang et al.; licensee BioMed Central. 2015
Received: 18 June 2014
Accepted: 21 November 2014
Published: 15 January 2015
Ticks are distributed worldwide and considered as vectors of many human diseases. Tick defensins, a family of antimicrobial peptides, form the first line of defense against pathogens.
A defensin-like gene, named Ds-defensin, was identified from a cDNA library of the hard tick Dermacentor silvarum collected from northeast China. The full-length cDNA of Ds-defensin was 225 bp, encoding a 74 amino acid peptide. The nucleotide sequence of Ds-defensin shared 98.2% similarity to putative defensin from Dermacentor marginatus. RT-PCR results suggested that Ds-defensin was extensively expressed in tick salivary gland and midgut, with a higher expression level in midgut. Ds-defensin showed broad antimicrobial activity against various Gram-positive and Gram-negative bacteria, as well as the fungus Candida albicans.
We characterized a functional defensin from D. silvarum of China. Ds-defensin showed bactericidal activity against various Gram-positive and Gram-negative bacteria. Ds-defensin can be expected to be introduced to the medical field as a new molecule with antibacterial activity.
KeywordsDefensin Antimicrobial peptide (AMP) Tick Dermacentor silvarum
Ticks, important medical arthropods, have great effects on animal and human health by transmitting various pathogens worldwide. Tick-borne pathogens include viruses, spirochetes, rickettsia, bacteria, and protozoa. They cause diseases such as tick-borne encephalitis, Crimean-Congo hemorrhagic fever, Lyme disease, Q fever, and Rocky Mountain spotted fever. In recent years, a new series of tick-borne diseases have posed a threat to the survival of mankind [1,2]. Many serological surveys also indicated the existence of populations with a variety of tick-borne infectious disease antibodies [3,4].
Hard ticks feed on mammals for a long time (several days), so that they have many opportunities to encounter microbes. Ticks do not have lymphocytes, thymuses, or antibodies. They rely heavily on antimicrobial peptides (AMPs) to defend against microbes so that they can live harmoniously with microbes [5,6]. AMPs are innate immune molecules that kill pathogenic microbes. Defensin is a well-known AMP in ticks. Defensins have been isolated from many species, including mammals, insects, and plants, and provide initial defense against infectious pathogens [7,8]. Several defensins and their isoforms have been identified in tick species including Ixodes scapularis, Amblyomma americanum, Dermacentor variabilis, Rhipicephalus microplus, Ornithodoros moubata, Ixodes ricinus, Amblyomma hebraeum and Haemaphysalis longicornis ticks [9–13]. Tick defensins usually contain six cysteine residues, and are usually expressed in the midgut (MG) after blood feeding or pathogen invasion [14,15]. Antimicrobial activity is primarily directed against Gram-positive bacteria, but some isoforms are also effective against Gram-negative bacteria, protozoa, and yeasts [9,15–17].
In our study, we characterized a defensin gene from a cDNA library of Dermacentor silvarum. This hard tick species was collected in the field of Heilongjiang Province, located in northeast China. It has been reported to transmit human pathogens, such as tick-borne encephalitis virus, Anaplasma, and Rickettsia [18–20].
Antimicrobial activity of Ds-defensin
Strain of microbes
Bacillus pumilus (CMCC63202)
Staphylococcus aureus (CMCC26003)
Micrococcus luteus (CMCC63202)
Mycobacterium bovis (carbenicillin-resistant)
Salmonella typhimurium (CVCC542)
Pseudomonas aeruginosa (CVCC2000)
Escherichia coli (CMCC44103)
Candida albicans (CAU0037)
Defensins are present in all types of organisms from humans and plants to arthropods. Defensins are AMPs that form the first line of defense against pathogens. A common function of defensins from all organisms is to lyse bacterial cells; however, the amino acid sequences of defensins show high diversity. Sequence analysis showed that the Ds-defensin peptide had 98.2% identity at the nucleotide level and 100% identity at the amino acid level to a putative defensin from D. marginatus, another hard tick species. Our data also show that defensins from hard and soft ticks shared a high degree of variability. The differentiation could be influenced by a diverse strategy of blood intake and types of pathogens that each tick species has encountered during their evolutionary history and geographical isolation .
The incidence of tick-borne diseases has steadily increased over the past few years, and effective vaccines against most tick-borne pathogens are currently unavailable . Defensin is an AMP that is not yet affected by antibiotic-resistance mechanisms . Defensins have been identified in both soft and hard ticks, and they show different profiles of antimicrobial activity. For example, Chrudimská and colleagues characterized two defensin isoforms (Def1 and Def2) from the hard tick I. ricinus, and showed that both Def1 and Def2 have significant bactericidal effects on Gram-positive bacteria but they are insensitive to Gram-negative bacteria, yeasts, and viruses . The same group recently characterized another defensin (defDM) from the hard tick D. marginatus, and suggested that defDM has inhibitory effects on Gram-positive bacteria and a popular tick-borne pathogen Borrelia afzelii . Lu et al. reported that a defensin-like peptide (DLP) from hard tick H. longicornis has potent antimicrobial activities against bacteria and fungi (C. albicans), and even shows strong antimicrobial ability against drug-resistant microorganisms . A recent review by Wang et al. summarized that the hard tick I. scapularis has two multigene families of DLPs . The core region of I. scapularis defensin scapularisin-20 exhibits a wide spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria, with higher potency to Gram-positive bacteria than to Gram-negative bacteria. The novel defensin we identified from the hard tick D. silvarum also has a distinct antimicrobial profile to different microbes. Ds-defensin showed strong potency against some Gram-positive bacteria (S. aureus, B. pumilus, M. luteus, and M. bovis), but had less effect on Gram-negative bacteria (S. typhimurium, P. aeruginosa, and E. coli). At a dose higher than 10 μM, Ds-defensin showed significant antifungal activity against C. albicans growth and could inhibit the antibiotic-resistant strain of M. bovis. Ds-defensin was not hemolytic at 20 μM, and had no detectable cytotoxicity against various human cells. These data suggest that Ds-defensin could be safely used in mammalian systems as a potential antimicrobial reagent against various Gram-positive bacteria and some fungi.
We thank Yikai Huang, Jiawei Tang, Tingting Li and Dan Cao for assistance with the experiments. This work was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions; National Natural Science Foundation of China (NSFC) (81172812, 81271792, 31200648, 31300714, 81471571 and 31400737), PostDoc Science Foundation (2013 M541725) and Jiangsu Natural Science Foundation (BK2012180). J.D. is a member of Jiangsu Provincial Innovative Research Team.
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