Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides

Peptides 32 (2011) 441–446

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Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides Houshuang Zhang, Wenjie Zhang, Xinzhi Wang, Yongzhi Zhou, Na Wang, Jinlin Zhou ∗ Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Minhang District, Shanghai 200241, China

a r t i c l e

i n f o

Article history: Received 19 October 2010 Received in revised form 1 December 2010 Accepted 1 December 2010 Available online 17 December 2010 Keywords: Rhipicephalus haemaphysaloides Salivary gland Antimicrobial peptide

a b s t r a c t The presence of an effective immune response in the hemocoel of ticks is crucial for survival, as it prevents the invasion of pathogens throughout the animal’s body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a subtraction hybridization cDNA library was constructed from the salivary glands of the unfed and fed female tick Rhipicephalus haemaphysaloides, and a novel cysteine-rich AMP designated Rhamp (R. haemaphysaloides antimicrobial peptide) was isolated and identified. The Rhamp was encoded by a gene with an open reading frame of 303 bp which encoded a mature peptide with 8 kDa molecular weight. No identity was found by BLAST search to any database entries. The sequence encoding the Rhamp was subcloned into the pGEX-4T vector and expressed in Escherichia coli. The recombinant protein of Rhamp showed chymotrypsin and elastaseinhibitory activity and markedly inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa, Salmonella typhimurium, and E. coli. Moreover, the recombinant protein also exerted low hemolytic activity. These results indicate the Rhamp is a novel antimicrobial peptide with proteinase activity from the tick R. haemaphysaloides. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Ticks encounter various microbes while sucking blood from an infected host and carrying these pathogens in themselves; therefore, they are important vectors of a wide variety of disease-causing bacteria, viruses, protozoa, and other pathogenic microorganisms [4,26]. Arthropods lack an adaptive immune response, while vertebrates have one, but arthropod vectors have an extensive spectrum of innate immunity mechanisms comprising different types of useful and important molecules which are essential for survival [2,11,16,17]. Innate immunity via antimicrobial factors is made up of a diverse protective immune system with options for arthropods to combat invading pathogens [7]. In the course of evolution, ticks have developed multiple antimicrobial factors because they encounter a large diversity of pathogenic microbes in their different life stages. Reports have indicated that ticks have the ability to control infections when challenged with various bacteria [19,28,29]. Many different compounds in the arthropod hemolymph have been shown to be involved in the recognition and elimination of invading microbes. Among these components, antimicrobial

Abbreviations: AMPs, antimicrobial peptides; Rhamp, Rhipicephalus haemaphysaloides antimicrobial peptide. ∗ Corresponding author. Tel.: +86 21 34293138; fax: +86 21 54081818. E-mail address: [email protected] (J. Zhou). 0196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2010.12.004

peptides (AMPs) play important roles; AMPs are mainly effective against Gram-positive and Gram-negative bacteria and also have potent activity against fungi, yeasts, and parasites [6,8,15,23]. Ticks can live harmoniously with microbes, mostly relying on AMPs for defense against microbes [41]. So far, a large number of AMPs have been found in different organisms, such as plants, fungi, mollusks, scorpions, amphibians, insects, birds, and mammals [7,10,30,35,36], and AMPs have become extremely effective and powerful weapons for invertebrate animals and plants to resist infection [6,8,12,15,24]. Recently, several antimicrobial peptides, including defensin-, ixosin-, and hebraein-like peptides, were identified from the hemolymph and salivary glands of ticks [18,20,21,34,39,46], and several small antimicrobial peptides, such as cecropin, defensin, maganin, and melittin, have been identified and reported to be very potent naturally occurring antibiotics [13,18,22]. Due to the growing problem of human pathogenic organisms which are resistant to conventional antibiotics, ticks are an interesting source of pharmacological substances to treat infections. Therefore, searching for more potent and effective antibiotics to combat pathogens resistant to conventionally used antibiotics is of great interest. Thus, knowledge of the antimicrobial peptides of Rhipicephalus haemaphysaloides is important for understanding the innate immunity in this vector tick and the role of this response in vector competence. The tick R. haemaphysaloides is a widespread tick species in China and other south Asian countries, and it transmits some

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significant diseases to animals and humans [44]. To date, there is no report on the identification of an antimicrobial peptide from this tick species. In this study, a novel antimicrobial peptide named Rhamp was identified and characterized from the tick R. haemaphysaloides. Result of BLAST search indicates that Rhamp does not have high identity with other known antimicrobial peptides. The results also showed that the recombinant Rhamp demonstrated antimicrobial activity against Gram-negative bacteria and possessed a strong inhibitory activity against two proteases, namely, chymotrypsin and elastase.

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as previously described [43]. The endotoxin of the purified proteins was removed using Detoxi-GelTM Endotoxin Removing Gel (Thermo Scientific, USA) according to the manufacturer’s instructions. The concentration of proteins was determined with a BCA protein assay kit (Thermo Scientific, USA). The proteins were sterilized by filtration through a 0.20 ␮m filter (Pall, USA) and stored at −80 ◦ C until use. Purified rRhamp was used for the subsequent experiments. 2.5. Antibacterial assay

2. Materials and methods 2.1. Ticks and tissue collection Ticks were maintained as previously described [45]. Briefly, the R. haemaphysaloides colonies were maintained in the laboratory by feeding on rabbits for several generations, and the rabbits were maintained in an isolated experimental animal facility. For tissue collection, adult female ticks were infested on the ears of rabbits. Ticks were recovered from the rabbit ears after 4 days, and the salivary glands were immediately dissected under the microscope [45]. The sample materials were stored at −80 ◦ C until use. 2.2. Subtractive hybridization and cDNA library construction for differentially expressed genes Total RNA was extracted using TRIzol (Life Technologies, USA) from 50 pairs of salivary glands dissected from partially fed female ticks or unfed female ticks. cDNA was synthesized using a SMARTTM PCR cDNA synthesis kit (Clontech, USA) according to the manufacturer’s instructions. A PCR-SelectTM cDNA Subtraction Kit (Clontech, USA) was used for cDNA subtractive hybridization to compare two populations of salivary gland mRNA from partially fed and unfed female ticks and to obtain clones of genes that are differentially expressed in these two populations. Finally, the PCR products were cloned into a pGEM-T Easy vector (Promega, USA). The salivary glands subtracted library (cDNA from partially fed female ticks as the tester and cDNA from unfed female tick as the driver) was constructed as previously described [47]. Randomly picked 2000 clones from these libraries were sequenced. 2.3. Cloning and analysis of the Rhamp gene The cloning and expression of Rhamp were performed as previously described [43]. A full-length Rhamp cDNA including the 5 - and 3 -end sequence was obtained with the SMART RACE cDNA Amplification Kit (Clontech, USA) according to the manufacturer’s instructions, and the clone obtained was sequenced on both strands. The secretory signal peptide was predicted by the SignalP server (http://www.cbs.dtu.dk/services/SignalP/). The cDNA fragment of Rhamp without a signal peptide was amplified by PCR using primers with the introduced EcoRI and XhoI sites (underlined), P1 (5 -GTGAATTCGAAAGAATATTGGACCTGAGAA-3 ) and P2 (5 -GGCTCGAGTAGCAGACACTCATGTTTTG-3 ), the AmpliTaq Gold Taq DNA polymerase (Applied Biosystems, USA) was used in PCR reaction mixture. The product was inserted into E. coli expression vector pGEX-4T-1 (Pharmacia Biotech, USA). The rRhamp fused with a glutathione S-transferase (GST) tag was expressed in the E. coli BL21 strain according to the manufacturer’s instructions. 2.4. Expression and purification of the recombinant Rhamp (rRhamp) The purification of soluble rRhamp was performed as described previously [42]. The purity of protein was determined by 15%

The antimicrobial activity of samples was monitored using liquid growth-inhibition assays [7]. The Poor medium (0.1% bactotryptone (Sigma, USA); 0.5% NaCl, w/v; pH 7.5) was used for bacterial cultures. Three strains of Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus, and Bacillus megaterium) and three strains of Gram-negative bacteria (Pseudomonas aeruginosa, Salmonella typhimurium, and E. coli) were used in this experiment. Briefly, bacteria were first grown in Luria–Bertani (LB) broth to an OD600 nm of 0.8. Bacterial suspension at a starting optical density at 600 nm (OD600) of 0.001 in the Poor medium was cultured in a sterile EP tube. A 1 mL bacterial culture was incubated with 20 ␮L of serial dilutions of purified antibacterial peptides filtered on a 0.22 ␮m Millipore filter. Twenty microlitres of a PBS solution and GST protein without peptide were added as the control. Bacterial growth was evaluated by measuring an increase of OD600 after incubation for 10 h at 37 ◦ C. Three independent experiments were performed for each sample. 2.6. Proteinase inhibition assays The purified peptide was pre-incubated with the concentrations for the following enzymes: chymotrypsin (1 ␮M), elastase (1 ␮M), and thrombin (1 ␮M) in a 100 mM Tris–HCl buffer, pH 8.0. These enzymes were pre-incubated with the rRhamp at a concentration range of 0.6–1.2 ␮M in a 100 mM Tris–HCl buffer, pH 8.0. After 30 min at 37 ◦ C, specific substrates were added: Nsuccinyl-Ala-Ala-Pro-Phe p-nitroanilide (1 mM) for chymotrypsin, N-succinyl-Ala-Ala-Ala-p-nitroanilide (1 mM) for elastase, and N-benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride (1 mM) for thrombin (Sigma, USA). The absorbance at 405 nm of reactions was determined using a spectrophotometer. Control reactions were performed under the same conditions but in the absence of peptide. The residual activity of enzymes was calculated. For the determination of the incubation time by Rhamp with proteases, proteases were incubated at equimolar ratios with rRhamp for variable periods of time before the addition of a substrate. The residual activities indicated by the amount of metabolized substrate were calculated. They were measured as absorbance values at 405 nm. Control reactions were performed under the same conditions using GST protein. The residual proteolytic activity (%) was determined comparatively. 2.7. Hemolysis assays Hemolysis assays were undertaken using rabbit red blood cells in a liquid medium as reported [3]. Briefly, fresh anticoagulated blood was collected from rabbits and washed twice with sterilized physiological saline (0.9% NaCl) by centrifugation at 1000 × g for 10 min. The resulting erythrocytes were then resuspended in saline (0.9% NaCl) to 4% (v/v). Serial dilutions of the peptide were used; 100 ␮L of an erythrocyte suspension were dispensed into 96-well plates in triplicate and incubated at 37 ◦ C for 1 h. Following centrifugation at 1000 × g for 5 min, the supernatants were transferred to new 96-well plates and monitored by measuring the absorbance at

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Fig. 1. Nucleotide and deduced amino acid sequences of cDNA encoding Rhamp. The cDNA has an open reading frame extending from position 15–317 and codes for 100 amino acid residues. The cleavage site of the Rhamp putative signal peptide is indicated by an arrowhead.

595 nm for released hemoglobin. Controls for 0% and 100% hemolysis consisted of cells suspended in saline and in 0.1% Triton X-100, respectively.

3. Results

3.3. Antibacterial activity The antibacterial activity of the peptide Rhamp was investigated against a variety of Gram-positive [G (+)] and Gram-negative [G (−)] bacteria. The results revealed that Rhamp exhibited antimicrobial activity against the G (−) tested strains (P. aeruginosa, S.

3.1. Construction of a cDNA library for differentially expressed genes The PCR-select cDNA subtractive hybridization method was applied to construct cDNA libraries for differentially expressed genes from the total RNA of tick R. haemaphysaloides salivary glands. After the transformation of E. coli cells, transformants were grown on agar plates without amplification in a liquid medium. The libraries were composed of approximately 2000 independent colonies. After sequencing, readable sequences of 1302 cDNA inserts were obtained. One of the EST sequences encoding the cysteine-rich protein was chosen for further analysis.

3.2. Identification and characterization of the Rhamp gene The Rhamp peptide was cloned and sequenced. The amino acid sequence of Rhamp deduced from the cDNA sequence is shown in Fig. 1; the full-length cDNA of Rhamp was 410 bp (GenBank accession number HQ337020), including a single open reading frame of 303 bp encoding a polypeptide of 100 amino acid residues, of which 25 residues were detected as a signal peptide. The analysis indicated that the Rhamp mature protein had an approximate molecular weight of 8 kDa and a pI of 8.82, as calculated with Genetyx software (Genetyx Inc., Tokyo, Japan). The Rhamp contained ten cysteine residues; thus, it is also a cysteine-rich antimicrobial peptide. Results of BLAST search indicate no high identity was observed to any entry in Genbank and the protein bank; however, a lower identity (28%, e-value: 0.073) to a cysteine-rich secreted protein of Ixodes scapularis (GenBank accession number XP 002433330) was observed. To characterize the Rhamp, rRhamp (without a signal peptide) was expressed in E. coli as a GST fusion protein. In SDS-PAGE analysis, the rRhamp protein migrated at approximately 34 kDa (Fig. 2).

Fig. 2. SDS-PAGE analysis of purified recombinant Rhamp. Purification of the rRhamp fused with GST was determined by 15% reducing SDS-PAGE. Purified recombinant proteins were subsequently used for the antibacterial, proteinase inhibition, and hemolysis assays.

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Escherichia

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Ti Time (H (Hrs)) Fig. 3. Antimicrobial activity of Rhamp. The Poor medium was used for bacterial cultures. Three strains of Gram-negative bacteria were used in this experiment. Bacterial growth was evaluated by measuring the increase in OD600 after incubation for 10 h at 37 ◦ C. The results are expressed as means of three individual experiments, and the error bars indicate the standard deviations. (*) The differences between the treatment with the GST protein and rRhamp fused with GST at the same protein concentration were significant (P < 0.05).

typhimurium, and E. coli). The growth of G (−) was significantly inhibited to about 50% at a maximum concentration of 0.5 ␮M (Fig. 3). Thus, purified Rhamp exerted its antimicrobial activities against G (−) bacteria but no effects on G (+) bacteria (S. aureus, M. luteus, and B. megaterium) (data not shown). 3.4. Proteinase inhibitory activity assay To determine whether Rhamp exhibited inhibitory activity against some selected proteinases, proteinase inhibition assays were carried out. The rRhamp inhibited proteinase activity at a concentration range of 0.6–1.2 ␮M and had maximum inhibitory activity at a concentration of 1.2 ␮M (Fig. 4A). In addition, Rhamp was found to inhibit about 90% of chymotrypsin and elastase activities within 4 min. Nonetheless, the peptide was unable to inhibit thrombin (Fig. 4B). 3.5. Hemolytic activity of the Rhamp peptide Rabbit red blood cells were used to check for hemolytic capability. The result showed that the Rhamp peptide had little hemolytic

activity against the erythrocytes of rabbit even with peptide concentrations up to 10 ␮M (data not shown). 4. Discussion Until now, hundreds of antimicrobial peptides have been isolated from various organisms. They are often short molecules (below 10 kDa) with a cationic character; however, most AMPs encompass a wide variety of structural motifs. Their differences in the primary structure result in a large difference in their antibacterial mechanisms [37] and mode of action [14]. Most AMPs of insect and arthropod conserve a characteristic motif of six cysteines, which form three disulphide bonds [7,20,21,27,39]. In this study, Rhamp, one novel cysteine-rich peptide with 8 kDa molecular weight, was isolated, identified, and biologically characterized from the hard tick R. haemaphysaloides. Sequence identity analysis showed that the Rhamp has a lower identity to a cysteine-rich secreted protein of I. scapularis. It is well known that some protease inhibitors are involved in several mechanisms of the immune system of arthropods; thus, this gene was studied as the target gene in the current study. 100

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Fig. 4. Proteinase inhibitory activity of rRhamp. (A) Residual activity of chymotrypsin, elastase, and thrombin in the presence of rRhamp at a concentration range of 0.6–1.2 ␮M. (B) Inhibition of proteinase by Rhamp as a function of the incubation time. Proteases were incubated at equimolar ratios with rRhamp for variable periods of time, as indicated on the x-axis before the addition of a substrate. Residual activities indicated by the amount of metabolized substrate are reported on the y-axis. They were measured as absorbance values at 405 nm. Control reactions were performed under the same conditions using the GST protein. The error bars indicate standard deviations. The residual proteolytic activity (%) was determined comparatively.

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However, the exact mechanism of the antimicrobial activity of AMPs is unknown. It remains unclear if the antimicrobial activity is due to a direct effect of the peptide on the bacterial membrane. There are several reports showing that AMPs may translocate through the bacterial membrane and act on intracellular targets, leading to inhibition of specific enzymes and macromolecule (DNA, RNA, protein, and cell wall) synthesis, as well as the alteration of cellular division [5,38]. In addition, researchers have demonstrated that anionic AMPs bind to the S. aureus membrane via interaction with a receptor [33] or that AMPs interact with metal ions, preventing their uptake by microorganisms [9]. A recent study revealed that AMPs may help overcome host defensive responses or keep the blood sterile in the tick body [25]. In mammals, AMPs are an important part of the innate immune system and may act either directly on microorganisms or by modulating the immune response (innate and adaptative) through (i) enhancement of phagocytosis, (ii) recruitment of immune cells to infection sites, (iii) induction of cytokines, and (iv) neutralization of the septic effects of lipopolysaccharides [31]. The large diversity of antimicrobial peptides suggests the antimicrobial mechanisms are more complex, and the discovery of Rhamp adds a new member to the increasing family of antimicrobial proteins/peptides, thus providing more evidence for the understanding of the antimicrobial mechanism. In addition, Rhamp showed proteinase inhibitory activity against chymotrypsin and elastase. Previous studies suggested that the pathogens use proteinases for: (i) invasion of the host tissues; (ii) acquisition of nutrients; and (iii) evasion of the host immune system. Thus, the presence of proteinase inhibitors may block the invasion and proliferation of pathogens [1]. Clarification is necessary to determine whether the Rhamp uses the same mechanism against microorganisms. In earlier studies, it was indicated that some AMPs have harmful side effects, such as hemolytic activities [12,32] or cytotoxicity toward mammalian host cells [40,41], which act as barriers for potential therapeutic agents. Therefore, the hemolytic activity and cytotoxicity of antimicrobial peptides are very important factors for evaluating their safety/toxicity. In this study, Rhamp exerted low hemolytic activity; thus, it can potentially be used as a therapeutic agent against bacteria. In conclusion, the tick Rhamp expressed in the salivary glands, was reported as a novel AMP. The novel molecule has antimicrobial activity against Gram-negative bacteria; thus, the Rhamp peptide could be developed as a future therapeutic agent against bacteria. Furthermore, it will be possible to extend this study to analyze the structural characteristics and mechanisms of antibacterial action in future research. Acknowledgements This work was supported by grants from the Basic Research Foundation for National Commonweal Institute of China (2006JB02) and the Foundation of State Key Laboratory of Veterinary Etiological Biology, China. References [1] Armstrong PB. The contribution of proteinase inhibitors to immune defense. Trends Immunol 2001;22:47–52. [2] Beier JC. Malaria parasite development in mosquitoes. Annu Rev Entomol 1998;43:519–43. [3] Bignami GS. A rapid and sensitive hemolysis neutralization assay for palytoxin. Toxicon 1993;31:817–20. [4] Bior AD, Essenberg RC, Sauer JR. Comparison of differentially expressed genes in the salivary glands of male ticks, Amblyomma americanum and Dermacentor andersoni. Insect Biochem Mol Biol 2002;32:645–55. [5] Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 2005;3:238–50. [6] Brown KL, Hancock RE. Cationic host defense (antimicrobial) peptides. Curr Opin Immunol 2006;18:24–30.

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