Identification and Molecular Characterization of Bioactive ...

TitleIdentification and Molecular Characterization of BioactiveMolecules Expressed in Salivary Glands of the HardTick,Haemaphysalis longicornis( 本文（FULLTEXT) )

Author(s) HARNNOI, Thasaneeya

Report No.(DoctoralDegree) 博士(獣医学) 乙第084号

Issue Date 2007-09-14

Type 博士論文

Version author

URL http://hdl.handle.net/20.500.12099/23174

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Identification and Molecular Characterization of Bioactive Molecules

Expressed in Salivary Glands of the Hard Tick, Haemaphysalis longicornis

（フタトゲチマダニの唾液腺に発現する生理活性分子の同定

および機能解明に関する研究）

2007

The United Graduate School of Veterinary Sciences, Gifu University

(Obihiro University of Agriculture and Veterinary Medicine)

HARNNOI, Thasaneeya

Identification and Molecular Characterization of Bioactive Molecules

Expressed in Salivary Glands of the Hard Tick, Haemaphysalis longicornis

（フタトゲチマダニの唾液腺に発現する生理活性分子の同定

および機能解明に関する研究）

HARNNOI, Thasaneeya

I

Contents

Contents I-II

Abbreviations III-V

General introduction 1-5

Chapter 1

Exploring bioactive molecules from salivary gland cDNA library of Haemaphysalis

longicornis

1-1. Introduction 6-8

1-2. Materials and Methods 8-11

1-3. Results 11-14

1-4. Discussion 14-17

1-5. Summary 17-18

Chapter 2

Molecular characterization and comparative study of 6 salivary gland

metalloproteases from the hard tick, Haemaphysalis longicornis

2-1. Introduction 26-28

2-2. Materials and Methods 28-35

2-3. Results 35-38

2-4. Discussion 38-42

2-5. Summary 42-43

II

Chapter 3

Identification of genes encoding cement-like antigens expressed in the salivary

glands of Haemaphysalis longicornis

3-1. Introduction 55-56

3-2. Materials and Methods 56-58

3-3. Results 58-60

3-4. Discussion 60-63

3-5. Summary 63-64

Chapter 4

Characterization of Haemaphysalis longicornis recombinant cement-like antigens

and evaluation of their vaccination effects

4-1. Introduction 69-71

4-2. Materials and Methods 71-76

4-3. Results 76-79

4-4. Discussion 80-83

4-5. Summary 83

General discussion 95-100

Conclusion 101-104

Acknowledgements 105-106

References 107-125

III

Abbreviations

A ADAM: a disintegrin and metalloprotease

B BCIP: 5-Bromo-4-chloro-3-indolyl Phosphate

C cDNA: complementary DNA

CIAP: calf intestinal phosphatase

C-terminal: carboxy terminal

D DAB: 3, 3’-Diaminobenzidine

DMSO: dimethylsulfoxide

DNA: deoxyribonucleic acid

E EDTA: ethylenediaminetetraacetic acid

EST: expressed sequence tag

G GST: glutathione S-transferase

H hlMP: H. longicornis metalloprotease

HRP: horseradish peroxidase

I IFAT: indirect fluorescent antibody test

Ig: immunoglobulin

IPTG: isopropyl-β-D(-)-thiogalactopyranoside

M MP: metalloprotease

Mr: marker

mRNA: messenger RNA

N NBT: nitroblue tetrazolium

N-terminal: amino terminal

NZY agar

IV

O ORF: open reading frame

P PBS: phosphate buffered saline

PCR: polymerase chain reaction

pfu: plaque forming unit

PI: propidium iodide

poly(A)+ RNA: polyadenylated RNA

PVDF: polyvinylidine difluoride

R RGD: Arginine-Glycine-Aspartate

RIM 36: Rhipicephalus immunodominant molecule 36

RNA: ribonucleic acid

RT: room temperature

RT-PCR: reverse-transcription-polymerase chain reaction

S SDS: sodium dodecyl sulfate

SDS-PAGE: SDS-polyacrylamide gel electrophoresis

SE: standard error

SVMP: snake venom metalloprotease

T TFPI: tissue factor pathway inhibitor

TAE: Tris-acetic acid-EDTA

TBE: Tris-Borate EDTA

TBS: Tris-buffered saline

X X-gal: 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside

V

Unit abbreviation

D oC: degree

H hr: hour

K kDa: kilo Dalton

kbp: kilo base pair

M M: mol/liter

μg: microgram

mg: milligram

min: minute

ml: milliliter

mmol: millimol

mM: milliM

N ng: nanogram

nm: nanometer

S s: second

μ μg: microgram

μl: microliter

1

General introduction

Ticks are blood feeding ectoparasite of mammals, birds and reptiles throughout

the world. Approximately 900 species have been described worldwide (9, 53). There

are two well established families of ticks, the Ixodidae (hard ticks), and Argasidae (soft

ticks). Both are important vectors of disease causing agents to humans and animals

throughout the world. A third family, the Nuttalielidae, contains only a single species.

Public health importance of ticks resides in their ability to transmit a greater variety of

infectious agents to humans and other animal species than any other blood-feeding

arthropods. They are also directly responsible for damage to hides and lost production

in livestock. The economic impact of ticks together with costs of control measures has

been estimated at between 14-18.7 billion USD globally in the livestock sector (23, 58).

Emerging tick-borne infectious agents include arboviruses, spotted fever group

rickettsias, ehrlichias, anaplasmas, and spirochetes (110, 124, 125). In addition, the

zoonotic potential of tick-transmitted disease of companion animals, livestock and

wildlife cannot be ignored (22).

Haemaphysalis longicornis Neumann, 1901 belonging to the Metastriata is

characterized by having both the parthenogenetic and the bisexual races (36, 52, 66).

As well as other ixodid ticks, H. longicornis has 4 stages including embryonated egg,

larva, nymph and adult. H. longicornis is distributed mainly in northeastern areas of

both Russia and China, the Korean peninsula, Japan, Australia, New Zealand, New

Caledonia and the Fiji Islands (52). This species is known as a vector of the rickettsia

causing Q-fever, viruses causing Russian spring-summer encephalistis and protozoa

causing theileriosis and babesiosis (10, 52). In Japan, H. longicornis is the most

2

abundant species in Japanese pastures that transmits bovine theileriosis caused by

Theileria bufferi/orientalis, bovine babesiosis caused by Babesia ovata among grazing

cattle (37, 38, 56) and canine babesiosis caused by Babesia gibsoni (51). Rodriguez

and coworkers also inferred the potential role of H. longicornis in the transmission of

equine babesiosis caused by Babesia cabali (99).

Ticks are pool feeder, ripping and tearing the delicate membranes and tiny

blood vessels of the dermis and sucking the fluids that are exuded into the wound.

Within 5-30 min, cement is secreted into the wound (109). This material harden quickly

into a latex-like covering around the mouth parts, which enable ticks to remain attached

to the host during the prolonged feeding period of 4-8 days and prevent host immune

response molecules from coming into contact with the tick proboscis (14). Cement

cone proteins represent candidates for inclusion in vaccine against ticks, or pathogen

they transmit, since the formation of the cone is essential for tick attachment and

feeding.

Tick feeding success is dependent upon the parasite’s ability to overcome three

components of the host defense system, namely (I) hemostasis, (II) inflammatory

response and (III) cell mediated immunity, especially the cell mediated cutaneous

basophil hypersensitivity (109). During probing for host blood, ticks cause physical

trauma to skin and small blood vessels will suffer extent of damage varying with the

tick species. The normal physiological response to disruption of blood vessels is

hemostasis, which involves coordinated interaction among platelets, coagulation

pathway proteins and endothelial cells (39). Ticks are able to prevent blood coagulation

at their feeding sites, ensuring continuous blood flow during the feeding period. Several

anticoagulant proteins from saliva have been described (71), some inhibit the extrinsic

3

and intrinsic pathway of blood coagulation (45, 70) and others inhibit platelet

aggregation (60, 62, 126). To deal with the inflammatory response, ticks secret several

anti-inflammatory and immunomodulatory components through their saliva such as

anticomplement or protease inhibitors (13, 69, 116). It is well known that ticks can

modulate both innate and acquired host immunity (129). Ticks are thought to have

evolved saliva that inhibits the cutaneous immune responses of their most common

host/s (92). Based on studies involving a variety of ixodid tick species and vertebrate

host species, elements of host immune defenses modulated by ticks include: inactivation

of complement components; diminished killing activity of NK cells; reduced antibody

responses to heterologous immunogens; inhibition of T-lymphocyte in vitro

proliferation induced by mitogens; suppression of production of pro-inflammatory

cytokines by macrophages; and suppression of Th1 cytokines concomitant with up-

regulation of Th2 cytokines (103).

Besides the essential role in blood feeding, tick saliva is also important in the

transmission of tick-borne pathogens (131). Some research has demonstrated that

components secreted from salivary glands can facilitate pathogen transmission to

vertebrate hosts (57). There is some evidence suggesting that animals exposed to tick

saliva were protected against parasite infections (11, 79). Factors that might affect

pathogen transmission and establishment in host repeatedly infested with pathogen-free

tick include alteration in the cutaneous environment at the tick attachment site, which

would interrupt feeding and/or be deleterious to introduced pathogens, reduction of

duration of attachment to the host (130). These evidences indicated that salivary protein

may be used as an immunogen to control pathogen transmission.

4

Control of tick infestations has been difficult because ticks have few natural

enemies. A major control component of integrated tick control methods is the use of

acaricides. However, application of acaricides has had limited efficacy in reducing tick

infestations and is often accompanied by serious drawbacks, including the selection of

acaricide-resistant ticks, environmental contamination and contamination of milk and

meat products with drug residues (46). Alternative approaches for tick control involve

the use of hosts with natural resistance to tick. Vaccines have been developed that

induce immunological protection of vertebrate hosts against tick infestation. Two main

approaches have been considered for tick vaccine development: the use of exposed

antigens and the use of concealed antigens (134, 140). Exposed antigens are tick

proteins injected into the host, which interact with the host defense system during the

course of tick feeding. Concealed antigens are hidden antigens that not exposed to the

host immune system. A vaccine for tick population control based on hidden gut antigen

has already been commercialized, but it is not boosted by tick challenge in the field

(135). Thus there is interest in the evaluation of salivary gland proteins that are exposed

to the host during tick feeding. Novel strategies are being sought to control tick and

tick-borne diseases including transmission-blocking vaccines that target tick saliva

proteins essential for pathogen transmission and/or establishment.

Success of host vaccination will depend on the identification, cloning and

expression of key physiological tick molecules. Recent advances in genomics and

proteomics studies on the transcriptmoes and proteomes of blood feeding arthropods

have provided important new insights into the complex pharmacology of vector saliva.

The discovery of tick salivary proteins has been greatly increased by novel molecular

biology techniques and bioinformatics analysis (95). Identification of the transcripts

5

and proteins present in the salivary gland of hard ticks such as Ixodes scapularis (97,

118), Rhipicephalus (Boophilus) microplus (101), Ixodes ricinus (117), Ixodes pacificus

(34), Amblyoma americanum (15) and Dermacentor andersoni (2, 15) have been

identified recently. Host-vector-pathogen interaction has been studied through the

identification of genes differentially expressed in salivary glands of female

Rhipicephalus appendiculatus infected with Theileria parva (81) and in I. ricinus and I.

scapularis salivary glands in response to the blood feeding (69, 143).

The identification of tick-protective antigens remains the limiting step in the

development of effective tick vaccines. Despite recent advances in the identification of

tick-protective antigens, few antigens have been tested as recombinant vaccines against

tick infestation (26). The aim of this study focused on identification, molecular cloning

and characterization of salivary gland proteins of H. longicornis for development of tick

control strategies. The objectives of this study are summarized as follows: (I) to

identify bioactive molecules or protective antigen that were expressed during blood

feeding by random sequencing of cDNA library from salivary glands of H. longicornis,

(II) to further characterize one selected bioactive molecule, metalloprotease that was

identified from the library, (III) to identify immunodominant antigens from cDNA

library by immunoscreening technique and (IV) to evaluate the vaccine potency of

immunoscreening positive clones.

6

Chapter 1

Exploring bioactive molecules from salivary gland cDNA library of

hard tick, Haemaphysalis longicornis

1-1. Introduction

Ticks are medically important vectors of human and animal diseases. They are

best known because of their ability to transmit a variety of pathogenic organisms,

including fungi, viruses, rickettsia, bacteria, and protozoa. Unlike most hematophagous

arthropods, which engorge rapidly, ticks remain on the host for a long time to complete

their blood feeding. Tick saliva contains a mixture of peptides and proteins serving a

variety of functions that are essential for creating and maintaining the blood pool or the

feeding lesion in host skin. Several bioactive molecules in tick saliva that affect the

host’s hemostatic, inflammatory, and immune system have been extensively studied

(93-95, 128). The proteins possessed anti-hemostatic activity including vasodilators,

inhibitors of platelet aggregation and blood-coagulation cascade have been identified

and characterized (30, 91, 116). Currently, post-transcriptional gene silencing using

RNA interference (RNAi) is a useful tool to elucidate the gene function. The first

application of RNAi in tick research has been demonstrated in a histamine binding

protein of A. americanum (3). By using RNAi for silencing gene encoding for salivary

anticoagulant, disruption of I. scapularis anticoagulation was demonstrated (78).

Ticks have also evolved a strategy to deal with the inflammatory response of the

host following tissue injury using several components such as anticomplement or

protease inhibitors (13, 69, 116). It is well known that ticks can modulate both innate

and acquired host immunity (129). Molecules in tick saliva, which possess

7

immunomodulatory activity that is important for survival and transmission of

pathogenic infectious agents (129) were identified (1). The characterization of a

recombinant immunomodulatory protein from the salivary glands of D. andersoni

suggested that p36 recombinant protein expressed in insect cells could suppress T-

lymphocyte-mitogen-driven in vitro proliferation of splenocytes from tick-naïve mice.

Saliva is also important in transmission of tick-borne pathogens, as it may enhance

pathogen transmission (131).

Currently, EST databases from salivary glands libraries of R. appendiculatus

uninfected and infected with T. parva were established (81). No major differences were

observed when these 2 libraries were compared, but there was evidence of up-regulation

of some proteins, such as a glycine-rich protein, in infected salivary glands. These data

provided information for the construction of a DNA microarray, which would be a

useful tool for probing vector-pathogen interactions.

The development of new technologies and approaches in the field of molecular

biology has shed light on the study of salivary molecules. The discovery of novel

proteins and genes expressed in salivary glands was accomplished by massive

sequencing of full-length cDNA libraries together with approaches involving

proteomics and functional genomics. A catalog of the transcripts and proteins from the

salivary glands of the malaria vector Anopheles gambiae has been published (31),

becoming an invaluable tool to study parasite-host and -vector interactions. The same

approach has been applied to identify a sialome of other vectors of disease (96, 119,

120). The same authors also studied the salivary compounds of I. scapularis by mass

sequencing of clones from salivary glands in the cDNA library (118). Random

sequencing of 735 clones yielded 410 cDNA clusters. One hundred clusters have been

8

identified as probable secretory products. Several novel sequences may have biological

activity and possibly serve as vaccine targets.

The hard tick, H. longicornis, which commonly infests cattle and dogs, is a

major vector that transmits pathogens such as Babesia, Theileria, and Borrelia to

domestic and wild animals in Japan and other East Asian countries (44). In this

research, the bioactive molecules produced from the salivary glands of the ticks are of

particular interest. A cDNA library from the salivary glands of H. longicornis was

constructed. Random sequencing of cDNA clones was used as a tool to recruit

biologically important key molecules that might be useful for the development of tick

control strategies.

1-2. Materials and Methods

Ticks

The parthenogenetic Okayama strain of H. longicornis (36) has been maintained

by feeding on rabbits and mice for several generations in our laboratory since 1997.

Extraction of total RNA and poly (A)+ RNA

Salivary glands from 160 adult ticks fed on rabbits for 3 days were homogenized

using a homogenizer (Ultra Turrax T8; IKA Labortechnik, Hohenstaufen, Germany) in a

Tri reagent (Sigma, St Louis, MO, U.S.A). The total RNA was subsequently isolated

according to the manufacturer’s instructions. Approximately 600 μg of total RNA was

obtained from 320 pairs of salivary glands. The poly (A)+ RNA was purified using an

Oligotex-dT30 (Super) mRNA isolation kit (Takara, Tokyo, Japan). The yield of

purified mRNA was 1% of the total RNA.

9

Construction of a cDNA library

The construction of the cDNA library was performed with 5 μg of mRNA using

a ZAP-cDNA® synthesis kit (Stratagene, CA, USA) according to the manufacturer’s

instructions. After size fractionation, the cDNA was ligated into an EcoRI/XhoI-ended

UniZap XR® vector. The ligation product was packaged using a ZAP-cDNA®

Gigapack® III packaging extract (Stratagene, CA, USA). Plating and titering of the

primary library was performed to determine the titer and ratio of the recombinant/non-

recombinant clones. The construction of the cDNA library yielded 1.65 X 105 pfu of the

primary library. The determination of the ratio of recombinant to non-recombinant

clones with the use of 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and

Isopropyl-1-thio-β-D-thiogalactopyranoside (IPTG) showed that the library contained

1.61 X 105 recombinant clones and 3,500 non-recombinant clones. Half the amount of

the primary library was used for further amplification. The amplified library with 4.0

X109 pfu/ml was aliquoted and kept at –85°C in 7% dimethylsulfoxide (DMSO).

Random sequencing of the H. longicornis cDNA library

The recombinant phage clones from the primary library were randomly selected

for sequencing. Four hundred and thirty-two white plaques were randomly selected for

PCR amplification. Two microliters of the phage suspension was used as a template for

PCR using T3 and T7 vector-specific primers flanking the cDNA inserts. The condition

of PCR was 30 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min,

and extension at 72°C for 2 min. The PCR products were analyzed by 1.5% Tris-Borate

EDTA (TBE) agarose gel electrophoresis. The clones containing large inserts were

selected for sequencing. After an in vivo excision experiment was performed, in order

10

to transfer the cDNA inserts from the lambda phage system to pBluescript phagemid,

plasmid DNA was further purified using a Qiagen mini kit for DNA sequencing.

Nucleotide sequences of cDNA fragments were determined using a BigDye terminator

cycle sequencing kit (PerkinElmer Life Sciences) and an automated sequencer (ABI

PRISM 310 Genetic Analyzer).

DNA sequence analysis

The sequences obtained by using vector-specific T3 and RPV primers were

analyzed. Primer and vector sequences were removed from raw sequences. The

nucleotide sequences were translated into amino acid sequences using an ExPASy

server (40) (http://www.expasy.org). The deduced amino acid sequences were

submitted to a similarity search against a non-redundant protein database at the National

Center for Biotechnology Information (NCBI) (at http://www.ncbi.nlm.nih.gov) using

the BLASTP program (7) and searched against the Conserved Domains Database

(CDD) (72) (http://www.ncbi.nlm.nih.gov/structure/cdd) DNA Databank of Japan

(DDBJ) (http://www.ddbj.nig.ac.jp/E-mail/homology.html). The sequences were next

analyzed for protein domain by using Pfam 12.0 (47) (Saint Louis) (at

http://pfam.wustl.edu). The FASTA files of the translated amino acid sequences were

also submitted to the SIGNALP server (82) (at http://www.cbs.dtu.dk/services/SignalP/)

to determine the presence of signal peptides. Multiple sequence alignment of nucleic

and amino acids was performed using ClustalW (85) at the EBML-EBI server

(http://www.ebi.ac.uk/clustalw/#) and edited using the Genedoc program (available at

http://www.psc.edu/biomed/genedoc). Conserved protein motifs were analyzed by

using a motif scan (28) (at http://myhits.isb-sib.ch/cgi-bin/motif_scan).

11

Nucleotide accession number

The nucleotide sequences reported in this thesis will appear in the

DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers

AB205028, AB205029, AB205030, and AB205031

1-3. Results

Random sequencing of the H. longicornis cDNA library

From 432 white plaques that have been randomly selected for PCR

amplification, 238 clones contained cDNA inserts with sizes ranging from 400 bp to 1.8

kbp. From 238 sequences studied, 80 had no possible open reading frame. These

clones were considered as junk sequences and were, therefore, excluded from further

analysis. The FASTA-formatted of all nucleotide sequences are available upon request

via email ([email protected]). Out of 158 sequences, 106 full-length cDNA

clones, which accounted for 67%, were obtained, as indicated by the presence of the

initiation codon. Sixty-five sequences were found to possibly contain the secretion

signal, whereas the secretion signal of 38 clones could not be determined since those

sequences were truncated.

A schematic display of cDNA sequence clusters from salivary gland proteins of

H. longicornis is shown in Fig. 1 and Fig. 2. Fig. 1 shows that, of the 158 cDNA

sequences studied, 43% match those related to Drosophila melanogastor and other

organisms. No significant similarity was found for 41% of the clusters, whereas only

16% gave significant hits with tick sequences. Fig. 2 shows that, of the 93 known

sequences, among the most common categories were housekeeping proteins including

ribosomal proteins (10%), mitochondrial enzymes (9%), hypothetical proteins (8%),

12

detoxification-associated proteins (3%), signal transduction-associated proteins (2%),

transcription/translation-associated proteins (1%), cytoskeletal proteins (1%), and

proteins that have been classified as “others,” which accounted for 38% of the total

sequences studied. Interestingly, 28% were coded for proteins that were probably

expressed during blood feeding.

There are 26 cDNA clones (28%) that possibly play an important role in the

blood-feeding mechanism. Twenty-three of them matched previously reported

sequences from ticks, and the remaining 3 sequences matched another organism. There

are 7 sequences that share similarity with the cDNA sequence isolated from H.

longicornis (accession number AB014612) (75). This protein, designated p29, was

shown to induce protective immunity against tick infestation in an experimental animal.

As shown in Fig. 3, the amino acid sequence of the p29 extracellular matrix of H.

longicornis was compared to 7 deduced amino acid sequences isolated from this cDNA

library. There were 3 sequences that gave the best match with H. longicornis HL35

antigen U protein (accession number AY550980) (115). The other 13 clones gave a

match to bioactive molecules involved in antihemostatic, immunosuppressant, and

secretory proteins with unknown functions. Three sequences matched other organisms

but possibly have similar functions to proteins in ticks, including metalloprotease and

dipeptidyl peptidase IV in Apis mellifera (honeybee), and the tissue factor pathway

inhibitor in Gallus gallus (red jungle fowl chicken). Multiple sequence alignment of

some of the deduced amino acid sequences of these cDNA was performed as shown in

Fig. 4 to 7. Mucins are large glycoproteins that generally have a high prolin, threonine,

and serine content. They are normally associated with mucus layers at the interface

between epithelia and their respective environment, particularly the lumens of hollow

13

organs of the vertebrate respiratory, digestive, and urogenital tracts (122). One cDNA

clone, designated H50 (accession number AB205029), gave the best match to the

mucin-like glycoprotein in agarisid tick, Ornithodoros moubata. The prediction of the

conserved domain using the Pfam server suggested that this protein contains a chitin-

binding domain. The clustal alignment of H50 (HLmucin) and similar proteins isolated

from other organisms is shown in Fig. 4. During the blood feeding stage, ticks secrete

some cement-like components to assist attachment to the host skin. Fig. 5 shows the

sequence alignment of the putative cement protein of R. appendiculatus, Rhipicephalus

immunodominant molecule 36 (RIM36) (17), which shares similarity with 2 cDNA

sequences isolated in this study and designated H184 and H416.

The tissue factor pathway inhibitor (TFPI) is a key molecule in the blood

coagulation pathway. In the present study, one cDNA sequence matched the TFPI

family. The alignment of H. longicornis TFPI and the first 2 best-matched species is

shown in Fig. 6 with the predicted Kunitz conserved domain.

Metalloproteases are other bioactive molecules of interest that possess anti-

hemostatic activity. The multiple sequence alignment of the metalloprotease enzymes is

shown in Fig. 7. The first sequence, called H135 (accession number AB205030),

matched I. scapularis MP1, which is similar to the hemorrhagic snake venom

metalloprotease of the reprolysin family, whereas the other sequence, called H292

(accession number AB205031), shared low similarity with the disintegrin-like protease

in A. mellifera (accession number XP_396339). Two other sequences, designated

H364 (accession number AB205028) and H410 matched the metalloprotease in I.

ricinus (69) and I. scapularis (32), respectively. The deduced amino acid sequence

showed that all sequences except clone H292 contained catalytic domain of reprolysin

14

family including catalytic domain (zinc-binding motif) metzincin (20)

(HEXXHXXGXXH), triad methionine (MSY) and a distally located methionine from

the first H of the zinc binding motif. On the contrary, H292 contains an ADAM

Cysteine-rich domain (ACR) located at the C-terminal of the protein (data not shown).

No significant similarity was found for 65 sequences. Multiple sequence

alignment of deduced amino acid sequences was performed via the ClustalW server

(data not shown). From the alignment score, 55% of the sequences appeared to be

multiple copies, whereas the remainders were single copies. From the prediction of

secretory signal peptide, it was found that 33 out of 65 possibly secreted. The

determination of the isoelectric point of these secretory proteins using an EMBL server

indicated that all of these proteins were basic in nature, with predicted pIs ranging from

8.1 to 12.2. The 12 most abundantly expressed sequences were further searched for a

conserved motif by using a motif-scanning server. It was found that most of the proteins

contained similar patterns of post-translational modification, such as the presence of a

N-myristolation site, a protein kinase C phosphorylation site, and a glycosaminoglycan-

attachment site. There were 6 cDNA sequences that encoded for the glycine-proline rich

protein, 5 sequences that were glycine-rich, and 3 sequences that were glycine-serine

rich. These 3 sequences also contained a multicopper oxidase signature and 20 copies of

a collagen triple helix repeat at the N-terminal part.

1-4. Discussion

A high-throughput approach designed to identify a large number of cDNAs was

used to obtain information on a variety of genes that are expressed in the salivary glands

of arthropods of medical importance (31, 95, 118-120). In the present study, a cDNA

15

expression library of salivary glands from the adult female tick H. longicornis was

constructed and randomly sequenced. The cDNA sequences can be categorized into 3

groups according to the results of blast search, namely, novel sequences for which no

significant similarity was found, sequences that matched previously reported proteins

from ticks, and sequences that matched other organisms.

Twenty-six clones possibly play important roles during blood feeding, and 7 of

these are identical to each other. Clustal alignment of these sequences revealed 39%

identity, suggesting that these cDNA sequences are similar but not identical to the

previously reported p29 sequences and can therefore be considered as a novel protein.

This p29 protein was described as a collagen-like protein that may be associated with

the formation of tick cement (75). Collagenous proteins belong to a multi-family, which

indicates that these proteins might share similar properties and functions to the p29

protein. The other 3 cDNA clones matched the H. longicornis HL35 antigen U protein.

Two sequences matched an unknown protein of Rhipicephalus haemaphysaloides, and 2

sequences matched the cement protein RIM 36 of R. appendiculatus (17). This protein

was shown to be present in salivary glands and the cement cone material secreted by the

tick. The data indicates that this cement component can induce strong antibody

responses in cattle and can therefore be a diagnostic marker for the detection of cattle

that have been exposed to R. appendiculatus ticks.

Calreticulin, a major calcium-binding protein of the endoplasmic reticulum,

appeared to be secreted in the saliva of A. americanum and Dermacentor variabilis.

Some evidence showed that an antibody raised against recombinant tick calreticulin

may be a biological marker of tick exposure (100). The cDNA sequence H186 was

shown to be identical to previously reported calreticulin in H. longicornis (142). The

16

biological function of this protein has not been clarified, although calreticulin may have

a role in angiogenesis in a feeding lesion.

Tick saliva contains pharmacologically active molecules that inhibit host

hemostasis, reduce pain, and modulate host inflammatory and immune responses. A

recombinant immunomodulatory protein from the salivary glands of D. andersoni has

been characterized (1) and shown to have immunosuppressive activity. One cDNA

sequence found in this library (H318) matched this protein, indicating a possible

function in counteraction with the host immune response. In the present study, 4 cDNAs

encoding for metalloprotease were isolated from a salivary gland cDNA library. Further

study of this cDNA sequence should be performed in order to elucidate the function and

biological activity of this bioactive molecule. In addition to its antihemostatic activities,

the metalloprotease enzyme also participates in angiogenesis, which is interesting for its

potential in wound healing and tissue remodeling. In this salivary gland library, one

cDNA sequence, which matched dipeptidyl peptidase IV (dPPIV), was found. This

molecule possesses angiogenesis activity. Characterization of these molecules may lead

to a better understanding of the biological process of tissue repair upon blood feeding.

The clone H50 matched with the mucin-like glycoprotein of O. moubata.

Mucins have been investigated as vaccine antigens. The protein was shown to induce

protective immunity in experimental animals (136). Though mostly isolated from

intestine, mucin-like glycoprotein was also reported from salivary glands of An.

gambiae (31). It is still not known whether the protein modulates parasite infectivity.

The evidence shows that this protein family is a promising candidate for a vaccine.

In this library, 41% of the sequence studied did not match any sequence reported

in the database. Analysis of the 12 most abundantly expressed sequences by using the

17

motif-scanning method suggested that these proteins contained some phosphorylation

sites. This indicated a possible involvement in signal transduction of some biological

process. Though the gene information or biological function still unknown, these

proteins secreted during blood feeding stage indicating some important role for tick to

take blood meal.

Herein, the results of the random sequencing of cDNAs from salivary glands

cDNA library of H. longicornis were reported. According to the characteristics and

possible functions of these proteins, including antihemostatic and immunodominant

ones or some that are involved in host-parasite interaction, further study will be required

to clarify their roles.

1-5. Summary

In tick salivary glands, numerous genes were induced during blood feeding in

order to counteract with host defense mechanism. The newly synthesized protein might

be important for the blood meal or be involved in host immune evasion and pathogen

transmission. In this study, cDNA library from salivary glands of partially fed adult

female H. longicornis was constructed and randomly sequenced, in an attempt to

identify genes that were expressed during blood feeding. The open reading frames of

158 cDNA clones were submitted to similarity search. A number of novel sequences

that gave no match to any sequence reported in the database were identified and

approximately 50% of these sequences are possibly secretory product. The majority of

known sequences are housekeeping genes. Several coding sequences possessed various

degree of homology to previously described proteins from other tick species. Several

genes encoding putative antihemostatic, immunomodulatory proteins and cement

18

proteins were found. This work provides information into the diversity of messages

expressed in the salivary glands of H. longicornis, describes novel sequences that may

be responsible for known biological activities, and identifies novel vaccine targets that

may be used in development of tick control.

19

Fig. 1. The BLASTP search results of cDNA sequences assigned as having no match to

the database or having hits to sequences obtained from ticks or other organisms.

Tick 16% (25)

Others 43% (68)

Novel 41% (65)

20

Fig. 2. The categorization of cDNA sequences according to their possible biological

functions.

signal transduction

transcription/translation

cytoskeletal detoxification

ribosomal protein

hypothetical mitochondrial enzyme

blood feeding

others

8 (9%)

26 (28%)

36 (38%)

7 (8%)

1 (1%) 3 (3%)

1 (1%)

9 (10%)

2 (2%)

21

Fig. 3. Clustal alignment of novel H. longicornis extracellular matrix proteins and

previously reported p29 protein (AB014612-1). Identical amino acids are shown in

black, whereas similar residues are shown in grey.

22

Fig. 4. Clustal alignment of novel H. longicornis mucin-like glycoprotein (H50) and

orthologous proteins from other organisms, including cabbage looper, Trichoplusia ni

(AAC47557), Aedes aegypti (AAM94146), Diamond-back moth, Plutella xylostella

(AAN63949), Anophleles gambiae (AAL68783), and O. moubata (AAS01023). The

predicted chitin-binding domain of HL mucin from the Pfam server is shown as a bar,

ranging from amino acid residue 83 to 138.

23

Fig. 5. Clustal alignment of the novel H. longicornis putative cement protein (H184,

H416) and the R. appendiculatus cement protein (RIM 36, AAK98794).

24

Fig. 6. Clustal aligment of the novel H. longicornis tissue factor pathway inhibitor

(H371), G. gallus TFPI (XP_421849), and Bos taurus TFPI (AAF61248). The Kunitz

conserved domain of HLTFPI, as predict by the Pfam server, is shown as a bar, ranging

from amino acid residue 122 to 174.

25

Fig. 7. Clustal alignment of the H. longicornis metalloprotease and previously reported

metalloprotease from I. scapularis MP1 (JC7969), MP2 (AAM93652), and I. ricinus.

MP (CAB55817). The conserved zinc-binding motif is shown in a box

(HEXXHXXGXXH). The methionine triad is shown as thick line.

HEXXHXXGXXH

26

Chapter 2

Molecular characterization and comparative study of 6 salivary gland

metalloproteases from the hard tick, Haemaphysalis longicornis

2-1. Introduction

As blood-pool feeders, ticks utilize a unique strategy to complete their long-

period blood meal (109). Their feeding mechanism is orchestrated initially by

penetrating their mouthparts into the host skin and damaging blood vessels for the

release of blood. During subsequent feeding, ticks secrete molecules through saliva,

which greatly contribute to establish and maintain the blood pool by interfering with the

host’s hemostatic, inflammatory, and immune systems (93-95, 128). Since the saliva

molecules are in direct contact with the hosts, a thorough understanding of their

functions is crucially important to the development of a tick vaccine.

Metalloproteases comprise a family of Zn2+-dependent enzymes that degrade

most components of the extracellular matrix (16). They are secreted in a proenzyme

latent form that requires activation for proteolytic activity. The ADAM (a disintegrin

and metalloprotease) family is characterized by the presence of disintegrin-like and

metalloprotease-like domains (141). Some ADAMs also displays additional domains

such as transmembrane and cytoplasmic domains and thrombospondin domains

characteristic for the ADAMTS (a disintegrin and metalloprotease with

thrombospondin) (29). The metalloprotease domain has the active site that contains zinc

atom and water molecules that are necessary for the hydrolytic processing of protein

substrates. Both the disintegrin-like domain and cysteine-rich domains have been

implicated in adhesive events such as binding to integrins and interacting with haeparan

27

sulfate proteoglycans, respectively (127). ADAMs are involved in diverse processes,

such as extracellular matrix degradation, development, cell-cell interaction, and cell

migration (104).

In ticks, genes encoding metalloproteases were identified from the saliva of I.

scapularis (118). These sequences are similar to snake venom metalloproteases

(SVMPs). SVMPs are members of the ADAMs family, whose main toxic effects are

due to the disruption of the hemostatic system. SVMPs are classified according to their

multidomain organization into class PI to PIV (48). The PI-class has only pre-

prodomain and metalloprotease domain, class PII has subsequence disintegrin domain,

class PIII has additional cysteine-rich domain and class PIV has the PIII domain

structure plus lectin-like domain connected by disulfide bonds. Tick metalloproteases

have molecular weight compatible with the PII-class (30 to 60 kDa) of SVMP, with

additional cysteine-rich domain (32). The tick metalloproteases have no Arginine-

Glycine-Aspartate (RGD) triplet typical for the disintegrin (118). The cysteine residue

spacing follows neither pattern found in disintegrins nor that found in the cysteine-rich

domain of the PIII-class (118). Such metalloproteases are similar to atrolysins, found in

snake venoms, which have fibrinogenolytic and gelatinase activities (48, 108). In

general, SVMPs display proteolytic activity against the basement membrane proteins,

which result in diapedesis of white blood cells (8, 18, 19, 105). As well as SVMPs, the

saliva of I. scapularis also possesses these metal-dependent anti-coagulant activities

(32). In addition, some types of snake venom also inhibit angiogenesis by the induction

of endothelial cell apoptosis (114). Similarly, it has been shown that late host response

to injury, such as endothelial cell-dependent wound healing in the blood feeding lesion,

is negatively modulated by tick saliva, and some evidence supports the notion that

28

metalloprotease is the enzyme that most likely accounts for this activity (33). In

conclusion, these studies suggest that metalloprotease in ticks is possibly essential for

inhibiting blood clotting and degrading extracellular matrix proteins for the preparation

of the feeding site and inhibiting host tissue repair in the late feeding phase via its anti-

angiogenic activity.

However, the biological characteristics of tick metalloproteases remain elusive,

although SVMPs have been extensively characterized due to their pathological

relevance and potential for therapeutic applications (88). In the present study, 6

metalloprotease genes from a salivary gland cDNA library of the hard tick, H.

longicornis were isolated and characterized. This is the first study to evaluate the

dynamic expressions in response to the blood feeding and the expression of the

recombinant protein and proenzyme activation mechanism of metalloproteases from the

salivary glands of ticks.

2-2. Materials and Methods

Ticks

The parthenogenetic Okayama strain of the H. longicornis tick has been

maintained by feeding on rabbits and mice for several generations in our laboratory

since 1997 (36).

Identification of metalloprotease genes in the salivary glands of H. longicornis

The cDNA library from the salivary glands of an adult female H. longicornis

was constructed using the vector-capping method (61, 147). The cDNA was

29

synthesized from 5 μg of total RNA by the G-C-capping method and ligated into the

plasmid vector pGCAP1. The EST database was constructed by randomly sequencing

10,000 recombinants, and 2 full-length metalloproteases designated as hlESTMP1 and

hlESTMP2 were isolated. On the other hand, 4 other metalloprotease cDNA sequences

herein referred to as hlMP1, hlMP2, hlMP3 and hlMP4 (referred to as H135, H292,

H364 and H410, respectively, in chapter 1) were isolated by random sequencing of 158

recombinants from a salivary gland cDNA library of the adult H. longicornis as

described in chapter 1. Since hlMP1, hlMP2, and hlMP4 appeared to be truncated,

identification of the 5’end terminus was performed by PCR amplification of the phage

library using T3 and gene-specific reverse primers (hlMP1:

5’GCCGAATTCGGACATTCC3’, hlMP2: 5’CTCAGGGCCAGTGGCTACT3’,

hlMP4: 5’ GATTTGGTCCAACTACACCT3’). The PCR reaction conditions were as

follows: 94°C for 10 min, 30 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 2 min,

and 1 cycle of 72°C for 7 min. DNA fragments with the expected size based on

multiple sequences alignment were excised from the gel, purified using Geneclean II kit

(Q-BIO gene®, Bio101 system, MP Biomedicals, CA, USA), and cloned into pGEM-T

Easy vector (Promega, Madison, WI, USA). Colony screening by PCR was performed

using gene-specific primers with 20 white colonies, and positive clones were selected

for sequencing. The analyses of the nucleotide and deduced amino acid sequences were

performed as previously described in chapter 1.

Phylogenetic analysis

A phylogenetic tree was constructed with the topology algorithms using

Genebee-Net (available at http://www.genebee.msu.su/services/phtree_reduced.html

30

server). The default method was used with PHYLIP and unrooted parameters. A

distance matrix method was used to create the tree with boostrap values of the full-

length metalloproteases.

Nucleotides accession numbers

The nucleotide sequences reported in this thesis will appear in the

DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers

AB264113, AB264114 and AB265817

Analysis of metalloprotease mRNA expression during blood feeding

To study the expression of H. longicornis metalloprotease (hlMP) genes during

blood feeding, 60 adult ticks were infested on a Japanese white rabbit, and 8 ticks were

collected every day until engorged (7 days). Ticks were dissected, and salivary glands

were pooled and kept in TRI® reagent (Sigma, St. Louis, MO, USA) at –80°C for

further RNA extraction. The expression patterns were studied by reverse transcription

PCR (RT-PCR) using gene specific primers (Table 1) and compared with those of

salivary glands from unfed ticks, as previously described in chapter 1. The RT-PCR

reaction conditions were as follows: 50°C for 30 min, 94°C for 2 min, 30 cycles of

94°C for 30 s, 50°C for 30 s, 72°C for 2 min, and 1 cycle of 72°C for 7 min. The H.

longicornis actin primers (accession number AY254898) were used as an internal

control for the RT-PCR reaction. To exclude the possibility of genomic DNA

contamination, negative-control reactions were performed without adding reverse-

transcriptase. The intensity of each RT-PCR product was quantified with the Macintosh

31

Luminous Imager version 2.0, and the values were expressed as a relative expression in

comparison to the actin gene. Data are expressed as means ± standard error of three

replicate experiments.

Expression of hlESTMP1 in E. coli

The PCR amplification of the pGCAP1 harboring the hlESTMP1 cDNA

sequence for the expression was performed using primers flanking the coding region of

that gene. A full-length hlESTMP1 containing a prodomain plus a metalloprotease

domain and a mature hlESTMP1 that contains only a metalloprotease domain were

subcloned into a BamHI restriction site of the pRSET-B expression vector (Invitrogen,

Carlsbad, CA). The full-length hlESTMP1 was amplified using the forward primer

(5’GAGGATCCGATGATTCTTCTTCTC3’) and the hlESTMP1 reverse primer

(5’GAGGATCCGTCATTTGTTTTGCCA3’). The start codon (ATG) and stop codon

(TCA) are indicated in bold. The BamHI restriction sites are indicated in italics. The

mature hlESTMP1 was amplified using the forward primer fused in-frame with the N-

terminal histidine tag gene (5’GAGGATCCGCGGCCAAATGTTCAA3’) and the same

reverse primer. The resulting plasmids were transformed into E. coli BL21 cell for the

expression of the recombinant hlESTMP1.

Purification of the recombinant hlESTMP1 fused with the histidine tag

The expression and refolding of the recombinant hlESTMP1 in E. coli were

performed as follows. Briefly, a single colony of E. coli BL21 harboring the

recombinant full-length or mature hlESTMP1 plasmids was grown overnight in 5 ml of

32

an LB-ampicillin broth. The culture diluted to 1:50 in 200 ml of a fresh LB-ampicillin

medium was grown until O.D600 reached 0.4. The isopropyl-beta-D-

thiogalactopyranoside (IPTG) induction was then performed with a final concentration

of 0.5 mM at 25°C overnight. For the purification of both recombinant proteins, the

inclusion bodies were resuspended with 10 ml of 6 M urea in an equilibration buffer

(100 mM NaH2PO4, 10 mM Tris-HCl pH 8.0). The mixture was incubated at 4°C for 2

hr and centrifuged at 15,000 X g for 20 min at 4°C. The supernatant was collected and

purified under a denaturation condition using Ni-NTA resin (Qiagen, Tokyo, Japan)

according to the manufacturer’s instructions. The proteins were eluted using the pH

gradient method. The refolding of the purified proteins was performed as described by

Ramos et al (87).

Production of an anti-hlESTMP1 serum

The full-length hlESTMP1 inclusion body was used to immunize ddy mice

(female, 6 weeks old) to obtain antiserum against hlESTMP1. The inclusion body was

washed for 6 times to remove the cellular proteins by sonicating in 1% triton-X-PBS

and one time in PBS. Immunization was performed for totally 4 times. First,

immunization was performed with an intraperitoneal injection of 100 μg protein in

Freund’s complete adjuvant (Sigma, St. Louis, MO, USA). The boosters were given at

2-week intervals with the same amount of protein in Freund’s incomplete adjuvant.

Blood samples were collected via a heart puncture after the third booster. The non-

specific antibodies were absorbed prior to use for Wester blotting and IFAT by

incubating antisera with lysate prepared from E. coli BL21 overnight at 4°C (100 μl

antisera: 50 μl E. coli lysate: 900 μl 5% skimmed milk in PBS).

33

Analysis of native hlMP1 in salivary glands by Western blotting

The salivary glands of unfed and 4-day fed female ticks were dissected out and

washed with cold PBS. The tissue was homogenized using a tissue homogenizer,

sonicated for 15 s, 3 times, and then centrifuged at 15,000 X g for 20 min at 4°C. The

supernatant was collected, and the protein concentration was measured using a Bio-Rad

Protein assay kit (Bio-Rad, Hercules, CA, USA). Ten micrograms of the soluble

fraction from the salivary gland was used as an antigen for sodium dodecyl sulfate poly

acrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The protein was

transferred onto a polyvinylidine difluoride (PVDF) membrane. The membrane was

reacted with mouse sera followed by goat anti-mouse-conjugated horseradish

peroxidase. The signal was detected by using a chemiluminescent substrate (Perkin

Elmer Life Sciences, Boston, MA, USA) following the manufacturer’s protocol.

Indirect immunofluorescent assay

The localization of native hlESTMP1 in salivary glands was detected by the

indirect immunofluorescent assay (IFAT). The whole salivary glands of unfed and 4-

day fed adults were placed on 14-well slides, air-dried, and fixed in acetone for 30 min.

The slides were further blocked with 0.5% skimmed milk in PBS for 1 hr at 37°C,

washed with phosphate-buffered saline (PBS), and incubated with anti-hlESTMP1

mouse serum diluted at 1:100 with 0.5% skimmed milk under the same conditions.

After washing, the slides were incubated with goat anti-mouse-conjugated alexa 488

diluted at 1:1,000 (Invitrogen). Nuclei staining were performed by incubating the slides

with 25 μg/ml propidium iodide (PI; Molecular Probe, Eugene, OR, USA) for 5 min at

34

room temperature and washing twice with PBS. The fluorescent signal was examined

with a confocal laser scanning microscope (TCS NT; Leica, Heiderberg, Germany).

Expression of hlESTMP1 in the baculovirus

The full-length hlESTMP1 with the signal peptide was amplified using primers

flanking the coding sequence (forward primer:

5’GCGGGATCCATGATTCTTCTTCTC3’, reverse primer:

5’GCGGGATCCTCAGTGATGGTGATGGTGATGTTTGTTTTGCCACCGATG3’).

The start codon (ATG) and stop codon (TCA) are indicated in bold. The BamHI

restriction sites are indicated in italics. The 6 histidine tag was added to a C-terminal to

aid purification. The PCR product was cloned into the BamHI restriction site of the

pFastBac Dual vector (Invitrogen). The recombinant donor plasmid was transposed to

the target site on the bacmid present in E. coli DH10BAC. Recombinant bacmid DNA

was prepared and used for transfection into Spodoptera frugiperda cells. The

recombinant baculovirus was amplified by multiple infections of insect cells. The viral

supernatant was used for the infection of High Five™ cells. After 5 days of infection,

the cells were harvested and washed with PBS. The cell pellet was resuspended in PBS

and lysed by sonication. The suspension was centrifuged at 10,000 X g, 10 min at 4°C

to separate the soluble fraction and cell debris fraction was utilized for SDS-PAGE and

Western blot analysis.

N-terminal sequencing of baculovirus-expressed hlESTMP1

The insoluble fraction of High Five™ infected cells was resuspended in a lysis

35

buffer (1% Nonidet P40 in PBS) and used for SDS-PAGE. Three major bands

corresponding to hlESTMP1, as proven by Western blotting using anti-hist tag

monoclonal antibody, were selected for sequencing. The protein was transferred onto a

PVDF membrane, and N terminal sequencing was performed by Edman degradation

(Hokkaido System Science Co., Ltd.).

2-3. Results

cDNAs cloning and sequences analyses of hlMPs

The PCR amplification of the 5’ terminus of 3 metalloproteases cDNA using a

phage library gave the multiple band products observed. The DNA fragments with

expected sizes were selected for sequencing. The full-length sequence of hlMP1 was

successfully obtained, whereas that of hlMP2 was unsuccessful due to the non-specific

amplification of the primers used. Identification of the full-length of the remaining

truncated cDNAs by using plaque hybridization and 5’ RACE failed to obtain the

missing 5’ terminus. The additional sequence of hlMP4 was obtained, but the starting

methionine was not identified.

It was found that the 6 hlMP proteins shared 13-41% identity to each other and

shared 16-42% identity with metalloproteases from other tick species. The multiple

sequence alignment of hlMPs with metalloproteases from other ticks is shown in Fig. 8.

Although the primary sequences shared a low percent identity, essential conserved

domains and critical residues were observed. These proteins were synthesized as a

proenzyme containing signal peptides, a prodomain, and a metalloprotease/cysteine-rich

domain. The amino acid sequences and conserved domain structures suggested that the

6 genes belong to the SVMP M12 family (EC 3.24.21) (90), with the catalytic site of the

36

metzincin subgroup (20). The phylogenetic analysis shown in Fig. 9 reveals the distinct

evolutionary patterns of these proteins. The hlESTMP1 is closely related to

Rhipicephalinae MPs. Except for hlMP1 that branch independently, the remaining

sequences, including hlESTMP2, hlMP2, hlMP3, and hlMP4, are close to each other.

Besides hlMP2, all the sequences studied possess a prodomain and a mature

metalloprotease domain. The structural features of each sequence are summarized in

Table 2. The hlMP3 contained an additional fragilysin domain that is similar to that of

the Bacteroides fragilis toxin (74), whereas hlMP2 contained only the ADAM/cysteine-

rich domain. All of the full-length sequences (hlESTMP1, hlESTMP2, and hlMP3)

contained a secretory signal peptide, as predicted by the SignalP server. The size of the

proteins and conserved domains suggested that hlMPs share common features with

snake venom class P-II except for the absence of the RGD triplet sequence of the

disintegrin domain.

The hlMPs have the zinc binding motif domain of the reprolysin family

(HELGHNLGXXHD), with some substitutions within the active site. The conserved

residues and motifs are summarized in Table 3. Besides hlMP2, the remaining 5

sequences possess all common characteristics of SVMPs, as described in I. scapularis

MPs, except for several amino acid substitutions of the reprolysin conserved domain.

The putative methionine turn of the triad MSY following the zinc-binding motif is

present in all 5 hlMPs. Three active site sequences, namely, hlESTMP1, hlESTMP2,

and hlMP3, have a G to A substitution as I. scapularis MPs, whereas hlMP4 remains

unchanged. The majority of substitutions are conservative except for hlMP3, which

changes from a hydrophobic amino acid to a polar one (L→T) and vice versa (N→V).

37

The expression of hlMPs mRNA transcripts during blood feeding

The expression profile of hlMPs during tick blood feeding is shown in Fig. 10

(a). The relative expression of hlMPs in comparison to the internal control actin based

on the band densitometry of RT-PCR products is shown in Fig. 10 (b). Accordingly,

hlMPs were strongly upregulated in salivary glands during blood feeding. Three

patterns of expression were observed (Fig. 10 a, b). Three genes, namely, hlESTMP2,

hlMP1, and hlMP2, were markedly induced when ticks started taking their blood meal,

gradually increased upon blood feeding, and decreased after ticks had fed near repletion.

In contrast, the expression of hlESTMP1 and hlMP4 was still relatively high even in

engorged ticks. Moreover, hlMP3 expression was apparently different from that of

other genes since the level of the transcript was markedly downregulated after the third

day of feeding.

Analysis of native protein and localization of hlESTMP1 in salivary glands

The heterologous expression of the full-length and mature hlESTMP1 in E. coli

was performed. The recombinant protein fused with a polyhistidine tag protein with an

expected size of 54 kDa and 36 kDa was obtained (data not shown). Western blotting of

salivary gland extracts showed that the antiserum against hlESTMP1 reacted strongly

with a protein of approximately 34 kDa from salivary gland extracts of partially fed

ticks and weakly reacted with that of unfed ticks (Fig. 11). The localization of

hlESTMP1 expression in salivary glands was shown by IFAT (Fig. 12). The fluorescent

signal was clearly detected in the cytoplasm of acini (mainly, type III acini) of the

glands of partially fed ticks reacted with antiserum against hlESTMP1 (Fig. 12 a).

38

Non-specific autofluorescence was observed in the chitinous basement membrane of the

gland acini reacted with normal mouse serum (Fig. 12 b).

Proenzyme activation and partial amino acid sequences analysis of baculuvirus-

expressed hlESTMP1

The full-length hlESTMP1 was expressed as a C-terminal hexahistidine tag

using a baculovirus expression system with the calculated size of 53 kDa (Fig. 13). The

majority of the recombinant protein was presented in an insoluble fraction. Western

blotting using a mouse anti-hist tag monoclonal antibody suggested that no recombinant

protein was secreted into the culture supernatant (data not shown). The recombinant

hlESTMP1 appeared to be processed into 2 smaller fragments with sizes of

approximately 38 and 26 kDa. These 3 bands are the processed forms of the

recombinant protein, as confirmed by N-terminal sequencing and Western blotting with

an anti-hist tag monoclonal antibody. The first band with molecular mass 53 kDa is the

full-length hlESTMP1 without signal peptide. The amino acid sequence of this

fragment, KEVSV (Fig. 13), corresponded to the signal peptidase cleavage site, as

predicted by SignalP (Fig. 13). The amino acid terminal sequences of two smaller bands

with predicted sizes of 38 and 26 kDa are APQVR and TYYSQ (Fig. 13), respectively.

The position of these cleavage sites are highlighted as red boxes in Fig.8.

2-4. Discussion

Identification and characterization of 6 metalloprotease genes in salivary

glands of H. longicornis were performed on the basis of sequence similarity and mRNA

39

expression patterns during blood feeding. The diverse functions of enzymes belonging

to this family, which included extracellular matrix degradation, inhibition of blood

coagulation (88), and inhibition of angiogenesis (145), permit a speculation on the

similar functions of these metalloproteases in ticks for the facilitation of the blood

feeding. Remarkably, the sequences of hlMPs showed unique and different features

when compared with those of SVMPs and I. scapularis MPs. Firstly, unlike SVMPs,

which contain a highly conserved prodomain that possibly resulted from gene

duplication (88), this region of hlMPs is quite diverse, indicating a different

evolutionary process. Secondly, several substitutions of active site residues were

observed in hlMPs, which apparently differ from I. scapularis MPs. The functional

significance of these substitutions remains unknown but possibly resulted in a high

degree of substrate preference. Since ticks feed on a variety of animals, the synthesis of

various metalloproteases may provide a large repertoire of tools to interact with animal

target proteins. Interestingly, these hlMPs also displayed different mRNA expression

patterns during feeding. Most hlMPs were constitutively expressed to different extents

before the ticks took blood meal. The subsequent feeding strongly triggered the

expression of all 6 genes. Remarkably, except for hlMP2 that showed slight expression

in ovaries, all of these genes were expressed only in salivary glands (data not shown).

This evidence may infer to the role of these metalloproteases around the host feeding

lesion, which might be involved in maintaining a feeding cavity and dealing with a late

host response to tissue injury, such as endothelial-cell-dependent wound healing.

Residue changes in the active site of hlESTMP1 are the subtlest among those

observed in other hlMPs. Therefore, hlESTMP1 possibly maintains its catalytic activity

or substrate specificity. Moreover, this gene is constitutively expressed and expected to

40

be involved in functions other than feeding. For these reasons, this gene was selected

for further characterization by expression as a recombinant protein. The recombinant

hlESTMP1 was used for the production of a specific antibody in mouse. The anti-

hlESTMP1 antibody reacted with the 34-kDa protein in the soluble fraction of the

salivary gland extract, which corresponded to the predicted size of the mature protein.

These results are in agreement with those obtained in I. scapularis MPs, in which the

mature form could be detected in the tick saliva (118). The full-length protein cannot be

detected even in unfed glands, indicating that the prodomain was intracellularly cleaved

after synthesis. As shown by IFAT, this protein is mainly localized in the salivary gland

cytoplasm of acini III, which is the main production site of several bioactive molecules

(95, 128), indicating that native hlESTMP1 might be synthesized and processed there

and released as the soluble mature metalloprotease into the acini lumen. The

simultaneous activation of the proenzyme suggested the existence of a natural specific

inhibitor of tick metalloproteases that regulate the activity of these mature enzymes.

The synthesis of proteases as proenzyme is one way to prevent the damage

caused by their actions in undesired locations on unintended substrates (68).

Understanding the proenzyme activation mechanisms would provide the necessary

information for the design of a selective inhibitor. In the case of ADAMs, it is likely

that the mechanism of maturation occurs via a pro-protein convertase-dependent

pathway. However, there are some cases in which ADAMs may undergo autocatalytic

activation (19). The autolysis mechanism was not observed in E. coli-expressed

hlESTMP1. The in vivo processing of the multidomain of hlESTMP1 was successfully

demonstrated using a baculovirus expression system. The full-length hlESTMP1 was

post-translationally modified into the mature domain in insect cells. Furthermore, the

41

N-terminal sequence of the recombinant hlESTMP1 suggests that the recombinant

enzyme was directed into the secretory pathway, where the signal peptide could be

cleaved by signal peptidase and the proenzyme was further processed into 2 smaller

fragments. The presence of 53 kDa fragment suggested the inefficient cleavage of the

prodomain by insect cells. It is still unknown why the third band with molecular mass

26 kDa is presented in the sample. This unexplainable phenomenon possibly happens

by the post-translational processing of insect cells that might not occur under

physiological condition of tick salivary glands. The most probable active form of the

baculovirus-expressed hlESTMP1 is the 38-kDa protein, since the amino acid APQVR

is in the position following the residue R or paired basic residue KK, reported cleavage

sites of furin-type proprotein convertase (12), indicating that the activation of this

proenzyme possibly follows this pathway. Post-transcriptional gene silencing, such as

the RNA interference technique targeting the enzyme responsible for the proenzyme

activation, would be a valuable tool to clarify this biological process.

Attempts to express both a full-length and truncated form of hlMPs as a GST

fusion protein failed to produce a soluble protein (data not shown). The insolubility of

recombinant hlESTMP1 hinders the characterization of its enzymatic activity. As in the

case of SVMPs expression, there have only been a few papers report the production of

this enzyme in a recombinant active form due to the large number of cysteine residues

in the disintegrin/cysteine rich domains (88). Searching for a proper expression or

refolding system will pave the way to clarify the role of these metalloprotease in ticks.

While most studies of tick-host interactions have focused on the mechanism of

anti-hemostasis, immune evasion and pathogen transmission, few studies have

concentrated on the effects of tick salivary gland molecules on wound healing (33, 60).

42

Most expression profiles of tick salivary glands have revealed a number of

metalloproteases (2, 32, 60, 118) with homology to those involved in extracellular

matrix (ECM) remodeling. The proteolytic environment created by tick-secreted

metalloproteases might be generating inhibitory peptides from ECM degradation that

contribute not only to delayed bite site healing and inhibition of blood coagulation but

also to immune evasion (2).

To this end, the integrated data obtained from this study could provide a novel

target for the development of a tick control strategy. This could be achieved by

disrupting the proenzyme activation mechanism to inhibit the production of an active

mature enzyme or by exploring metalloprotease regulators, such as inhibitors or

blockers of enzyme function, by vaccinating host animals with this bioactive molecule.

2-4. Summary

Genes encoding metalloproteases were identified from salivary gland cDNA

library of H. longicornis. The proteins are similar to snake venom metalloprotease of

the reprolysin family. The H. longicornis metalloproteases are proteins containing pre-

and prodomains, the zinc binding motif HEXXHXXGXXH common to the

metalloprotease and a cystein-rich region. Among these genes, apparent differences in

evolution and gene regulation during blood feeding were observed. Interestingly,

several amino acid substitutions within the active site of catalytic metalloprotease

domain were also observed. Molecular cloning and expression as a recombinant protein

of one selected gene was performed. This provided interesting data concerning tissue

localization and native protein size of this molecule in salivary gland. Moreover,

mechanism of proenzyme activation to produce a mature active enzyme was speculated.

43

These integrated information provided a better understanding of the role of this enzyme

in tick feeding mechanism.

Table 1. The gene specific primers used for RT-PCR in this study.

44

Primer Nucleotide (5’-3’)

hlESTMP1 forward GCGGAACTCATTGCTTATATGG

hlESTMP1 reverse GCAGTACACCCCCATGTAGGAC

hlESTMP2 forward GCGGGATCCGTTGAAGTATTTATA

hlESTMP2 reverse GCGGGATCCTCTCATTGTCTCCGACG

hlMP1 forward GGCACTGAA GCAGTAGCACTT3’

hlMP1 reverse GCGGGATCCCTATTCTTGTACACA

hlMP2 forward GCTTCTGAAAGGAGTGGAGC

hlMP2 reverse GTTCCAACTCTGACTGCAATTTCTCC

hlMP3 forward CGCCACAAGTTCACCAAGAA

hlMP3 reverse CATTTCATGTAGTCCACGGATT

hlMP4 forward CCGGCTCACGCTTGTCTT

hlMP4 reverse GATTTGGTCCAACTACACCT

hlactin forward GGTTGCCGCCCTGGTGGTTGA

hlactin reverse GCCGCACGATTCCATACCCAGG

45

Tabl

e 2.

Stru

ctur

al fe

atur

es o

f hlM

Ps

Sequ

ence

Id

Nuc

leot

ide

acce

ssio

n

num

ber

Nuc

leot

ide

(bp)

Am

ino

acid

aa

(kD

a)

Sign

al

pept

ide

aa (k

Da)

Prod

omai

n

aa (k

Da)

Met

allo

prot

ease

/cys

tein

e-ri

ch

dom

ain

aa (k

Da)

AD

AM

/

cyst

eine

-ric

h

dom

ain

aa (k

Da)

Frag

ilysi

n

dom

ain

aa (k

Da)

hlES

TMP1

A

B26

4113

15

93

483

(54.

8)

17 (1

.7)

185

(20.

8)

298

(34.

0)

- -

hlES

TMP2

A

B26

4114

17

71

512

(57.

5)

16 (1

.6)

174

(19.

8)

376

(41.

9)

- -

hlM

P1

AB

2050

30

1575

48

2

(55.

1)

25 (2

.7)

180

(20.

6)

303

(34.

6)

- -

hlM

P2

AB

2050

31

1090

27

4

(30.

7)

- -

- 27

4 (3

0.7)

-

hlM

P3

AB

2050

28

1708

50

6

(56.

8)

16 (1

.8)

171

(19.

3)

335

(37.

5)

- 10

2 (1

1.0)

hlM

P4

AB

2658

17

1193

39

7

(45.

9)

- -

250

(29.

0)

- -

46

Table 3. Conserved residues and motifs of hlMPs

Sequence Id Metzincin HELGHNLGXXHD MSY No. of

cysteine

hlESTMP1 3 HELAHSLGAEHD 3 13

hlESTMP2 3 HEVAHTLGATHD 3 15

hlMP1 3 HEIAHSFGCVHD 3 15

hlMP2 5 LQVALLLNASRD 5 12

hlMP3 3 HETAHVLGSFHD 3 11

hlMP4 3 HEMGHTLGCSHD 3 7

Metzincin is characterized by the presence of the triad MSY and conserved Zn2+

binding motif (HEXXHXXDXXH). The amino acid sequences of the reprolysin family

are shown (HELGHNLGXXHD). The substitutions of residues in the active site of

hlMPs are shown in bold.

47

Metalloprotease-cysteine rich domain

Prodomain

48

Fig. 8. Multiple sequence alignments of hlMPs with other tick MPs.

The CLUSTAL alignments of 6 hlMPs including hlESTMP1 (AB264113), hlESTMP2

(AB264114), hlMP1 (AB205030), hlMP2 (AB205031), hlMP3 (AB205028), hlMP4

with R. haemaphyloides (ABD66751), R. (B.) microplus, (AAZ39657) I. ricinus

(CAB55817), I. scapularis MP1 (JC7969), I. scapularis MP2 (AAM93652) and I.

scapularis MP3 (AAM93653) are shown. The predicted signal peptides are underlined.

The arrows indicate above the aligned sequences are prodomain and metalloprotease-

cysteine rich domain of hlMPs, as predicted by conserved domain database. The black

box indicates active site. The closed triangles denote cysteine residues. The triad MSY

is underlined by thick line. The identity (black shadow), highly conserved (dark grey

shadow) and conserved residues (light grey shadow) are marked. The red boxes

indicate N-terminal sequences of processed baculovirus expressed hlESTMP1, KEVSV,

APQVR and TYYSQ.

49

Fig. 9. Phylogenetic analysis of hlMPs with other tick MPs.

The phlylogenic analysis of hlMPs with R. haemaphyloides, R. (B.) microplus, I. ricinus,

I. scapularis MP1, I. scapularis MP2, I. scapularis MP3. The bootstrap values are

shown on the lineage of the tree.

R. (B.) microplus

hlESTMP1

I. scapularis MP2

R. haemaphysaloides

I. scapularis MP3

I. ricinus

I. scapularis MP1

100

97

100 hlESTMP2

100

hlMP3

100

100

hlMP4

hlMP2

100

100

100

92

hlMP1

50

Fig. 10. The expression analyses of hlMPs mRNA in pooled salivary glands of female

ticks fed on rabbits.

a) The mRNA expression using RT-PCR of hlESTMP1, hlESTMP2, hlMP1, hlMP2,

hlMP3 and hlMP4 in salivary glands from unfed, 1 day fed (day1), 3 day fed (day3), 5

days fed (day5) and engorged ticks. The amplification of actin gene (hlactin) was use as

an internal control.

hlESTMP1 0.4

0.3

hlMP1 0.5 0.4

hlMP2 0.1 0.2

hlMP3 0.3 0.2

hlMP4 0.4 0.3

hlactin0.5 0.4

Unfed Day1 Day3 Day5 engorged

hlESTMP2 1.0 1.5

Kbp

a)

51

b) The relative expression of hlMPs in comparison to actin gene.

hlESTMP2

hlESTMP1

hlMP2

hlMP1

hlMP3

hlMP4

b)

0.000

0.200

0.400

0.600

0.800

1.000

1.200

unfe

d

Day1

Day3

Day5

engo

rged

rela

tive e

xp

ress

ion

0.0000.2000.4000.6000.8001.0001.200

unfe

d

Day1

Day3

Day5

engo

rged

rela

tive e

xp

ress

ion

0.000

0.200

0.400

0.600

0.800

1.000

1.200

unfe

d

Day1

Day3

Day5

engo

rged

rela

tive e

xp

ress

ion

0.0000.2000.4000.6000.8001.0001.200

unfe

d

Day1

Day3

Day5

engo

rged

rela

tive e

xp

ress

ion

0.0000.2000.4000.6000.8001.0001.200

unfe

d

Day1

Day3

Day5

engo

rged

rela

tive e

xp

ress

ion

0.0000.2000.4000.6000.8001.0001.200

unfe

d

Day1

Day3

Day5

engo

rged

rela

tive e

xp

ress

ion

52

Fig. 11. Western blot analysis of salivary gland antigen extract with anti-hlESTMP1.

Western blotting of salivary gland extract of unfed (lanes 1 and 3) and 4 days fed ticks

(lanes 2 and 4) reacted with non-immune mouse serum (lanes 1 and 2), mouse

antiserum against hlESTMP1 (lanes 3 and 4). Mr, pre-stained marker. Arrow indicates

the size of native mature hlESTMP1.

kDa Mr 1 2 3 4

98

22 16

64

50

36

148

34 kDa

53

Fig. 12. The localization of hlESTMP1 in salivary glands of partially fed ticks by IFAT.

The cytoplasm of salivary glands acini, mainly acini III, from partially fed ticks

specifically reacted with anti-hlESTMP1 sera (green), but not with granules (a). Non-

specific autofluorescence was observed in the chitinous basement membrane of the

gland acini reacted with non-immune mouse serum (b). PI (red nuclei staining by

propidium iodide).

Anti-hlESTMP1 PI Merge

Non-immune mouse serum PI Merge

(a)

(b)

54

Fig. 13. The baculovirus-expressed hlESTMP1 and amino terminal sequences of the

cleavage sites. The amido black staining of hlESTMP1 expressed by baculovirus on

PVDF membrane (lane 1) and Western blotting of the same sample reacted with mouse

anti-HIS tag monoclonal antibody (lane 2). Mr, pre-stained marker. The corresponding

amino terminal sequences are shown as KEVSV, APQVR and TYYSQ.

Mr 1 2

98

22

16

64

50

36

148

KEVSV

APQVR

TYYSQ

N-terminal sequence

55

Chapter 3

Identification of genes encoding cement-like antigens expressed in the

salivary glands of Haemaphysalis longicornis

3-1. Introduction

Ticks are medically important vectors of human and animal diseases. In

addition to transmitting pathogens to mankind, tick infestations in animals produce

worldwide economic losses (23). Although tick control strategies rely extensively on

the use of chemical acaricides, this type of control is becoming less sustainable due to

the development of resistance against acaricides among ticks and the undesirable

contamination of the environment and animal food products (137). Therefore, the

development of alternative control strategies is necessary, in which vaccination against

ticks is a candidate method. A concealed antigen based-vaccine against R. (B.)

microplus (54) infestation was successfully commercialized (135). A concealed antigen

in the tick gut, however, is unlikely for the boost of the immune response by natural tick

exposure compared with salivary glands antigens.

As a vaccine target, salivary glands are a rich source of exposed antigens that

can stimulate host immune responses following natural infestation. Tick saliva contains

a mixture of peptides and proteins serving various functions that are essential for the

creation and maintenance of the blood pool or the lesion in the host skin for feeding.

Several bioactive molecules in tick saliva that affect the host’s hemostatic, inflammatory,

and immune systems have been extensively studied (93-95, 128). The discovery of

novel proteins and genes expressed in salivary glands has been accomplished by

massive sequencing of full-length cDNA libraries together with approaches involving

56

proteomics and functional genomics. This information assists in the exploration of

protective antigens for vaccine development.

Several vaccine candidates have been studied and tested for vaccine potency

against H. longicornis infestation (55, 75, 115). However, the animals vaccinated with

these molecules conferred partial protection against not all stages of tick. The

identification of other candidates for use in a cocktail vaccine is still required.

In this research, of particular interest are the immunogens produced from the

salivary glands of ticks. A cDNA library from the salivary glands of H. longicornis

would provide valuable material, as it would contain a wide variety of genes encoding

putative vaccine candidates. The immunoscreening method was used to recruit

immunodominant key molecules that might be useful for the tick control strategy.

3-2. Materials And Methods

Ticks

The adults and nymphs of parthenogenetic Okayama strain of H. longicornis

maintained by feeding on rabbits and mice for several generations in our laboratory

since 1997 were used in this experiment (36).

Animals

All animal experiments were conducted in accordance with the Standards

Relating to the Care and Management of Experimental Animals promulgated by

Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan

(Allowance No. 17-77).

57

Immunoscreening of the cDNA library

The cDNA library of salivary glands from adult H. longicornis was obtained, as

previously described in chapter 1. Approximately 20,000 pfu per plate of the amplified

library was used for plaque lifting. The library was grown on NZY agar for 3.5-4.0 hrs

at 42°C. Then, IPTG induction was performed at 37°C for 3.5 hrs by placing

nitrocellulose membranes soaked with 10 mM IPTG onto the surface of the agar. For

secondary and tertiary screening, 600 and 200 pfu were used for plating, respectively.

The total of 5 X 105 pfu of the cDNA library was screened using a polyclonal rabbit

anti-H. longicornis tick immune serum prepared as previously described (144).

Immunoscreening was performed using a Picoblue TM immunoscreening kit (Stratagene,

CA, U.S.A), according to the manufacturer’s instructions. After blocking a non-specific

binding site with 1% bovine serum albumin (BSA) for 1 hr, the membranes were

allowed to react with polyclonal rabbit anti-tick saliva serum diluted 1:200 in 1% BSA

in Tris-Buffered Saline (TBS) for 3 hrs. After washing 3 times with Tris-Buffered

Saline and 0.05% Tween 20 (TBS-T), the membranes were further incubated with

alkaline phosphatase-conjugated goat anti-rabbit IgG at a dilution of 1:10,000. The

Nitroblue Tetrazolium (NBT) and 5-Bromo-4-chloro-3-indolyl Phosphate (BCIP)

substrates were used for the visualization of the positive signals. The positive plaques

were subjected to further secondary and tertiary screening until the isolated clones were

obtained. The cDNA inserts of the positive clone were then excised from the lambda

phages to obtain the plasmid DNA for sequencing.

Nucleotides accession numbers

The DNA sequences analysis was performed as described in chapter 1. The

58

nucleotide sequences reported in this thesis will appear in the DDBJ/EMBL/GenBank

nucleotide sequence databases with the accession numbers AB252633, AB259292,

AB259293, AB259294, AB259295, AB259296, AB259297, AB259298 and AB259299.

RT-PCR analysis of mRNA expression in salivary glands

The expression of immunodominant mRNA transcripts in the salivary glands of

H. longicornis was studied. RT-PCR was performed using a TAKARA one-step RNA

PCR kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions. For the

PCR amplification of the total RNA from the salivary glands of unfed and partially fed

ticks, the specific primers of each cDNA clone were used. The primers specific for the

H. longicornis actin gene were used as an internal control for the RT-PCR reaction. A

negative-control reaction was simultaneously performed under the condition without

adding reverse-transcriptase to exclude some contamination of genomic DNA.

3-3. Results

The amplified salivary gland cDNA library was screened with polyclonal rabbit

anti-H. longicornis tick immune serum. Screening of 5 X 105 pfu yielded 17 positive

clones. PCR amplification showed that positive clones contained inserts with sizes

ranging from 0.5 to 1.7 kbp. As shown in Table 4, the BLASTP search indicated that 8

amino acid sequences were novel proteins; 8 matched with hypothetical proteins in H.

longicornis; and one with the H. longicornis HL35 antigen U (115). Ten of 17

sequences were found to possibly contain a secretory signal peptide. The remaining 7

sequences appeared to be truncated since starting methionine or 5’UTR were not found

(marked as “T”). Multiple amino acid sequence alignments of 8 novel proteins are

59

shown in Fig. 14. These sequences shared 23-97% identity to each other. Three

sequences, designated hlim10, hlim11, and hlim20, shared greater than 90% identity

were, therefore, considered to be the same. The amino acid sequence alignments

suggested that these proteins would have the unique features of cement proteins

contained in tick saliva (17), as indicated by the presence of several GL[G/Y/S/L/F]

tripeptide repeats.

One sequence, designated hlim3 (accession number AB252633), matched with

the previously reported HL35 (115). This sequence also possessed the characteristics of

a cement component (17). The multiple sequence alignments of hlim3, HL35

(accession number BAB20588), and its homologue, HL34 (accession number

BAB1376), are shown in Fig. 15. Even though the amino acid sequence of hlim3

shared greater than 80% identity with HL35, several amino acid substitutions and

insertions were observed, suggesting the presence of sequence polymorphism.

The remaining identical 8 sequences shared weak similarity with the

hypothetical protein of H. longicornis (77). The calculated molecular weight was

approximately 12 kDa and acidic in nature. The prediction from the SIGNALP server

suggested that the sequence contained a secretory signal peptide.

The expression of the mRNA transcripts of immunoscreening-positive clones

in the salivary glands of unfed and partially fed ticks was studied using RT-PCR (Fig.

16). Based on the amino acid sequence identity, the clones can be arbitrarily grouped

into 8 categories, namely, 6 novel sequences (5 singletrons and one tripletron), the H.

longicornis hypothetical protein, and the HL35 homologue. The results suggested that

all 8 sequences studied were expressed in the salivary glands of partially fed ticks and 7

sequences, namely hlim1, hlim2, hlim4 (faint band), hlim13, hlim14 (faint band), hlim20,

60

and hlim21, were also expressed in unfed ticks. No obvious difference in the expression

level of 4 transcripts (hlim2, hlim13, hlim20, and hlim21) between RNA of unfed and

fed ticks could be observed, whereas the remaining 4 (hlim1, hlim3, hlim4, and hlim14)

appeared to be upregulated during blood feeding.

3-4. Discussion

The cloning and expression of several key molecules for use as tick vaccine

candidates are ongoing worldwide. The study on antigens that elicit an antibody

response is a useful strategy to identify vaccine antigens. For this purpose, the cDNA

expression library of salivary glands from the adult female tick H. longicornis was

constructed in the present study. The genes encoding for immunodominant proteins

were identified by immunoscreening using rabbit sera after repeated infestation with H.

longicornis (144). The BLASTP search of 17 positive clones revealed that 8 clones did

not yield any similarities with known proteins, 8 clones gave matches with hypothetical

proteins of H. longicornis and one clone, hlim3, shared similarity with the HL35

antigen U. The vaccination effect of HL34, the homologue of HL 35, has been

evaluated, suggesting its suitability as a tick vaccine candidate (115). The clone hlim3

shared 83% identity with the HL35 protein; however, the substitution and insertion of

several amino acids were observed from the sequence alignments, indicating the

presence of gene polymorphism.

Eight cDNA clones gave best matches with the hypothetical protein from H.

longicornis, indicating that this gene was abundantly expressed. The RT-PCR results

showed that the mRNA transcript was upregulated in salivary glands during blood

feeding. The peptides are possibly related to a secretory product, as they display a

61

secretion signal. These findings implied that this protein plays an important role during

blood feeding, although its function is still unknown.

The deduced amino acid sequences of the 8 novel protein possessed striking

feature of cement substances even though no hits to any sequence reported in the

database were obtained (17). Further localization study by immuno-staining

experiments using specific antiserum that detect the proteins at the feeding site would

confirm the possible function of these proteins. The presence of glycine triplet repeats

and the high content of glycine are characteristic features of the vertebrate extracellular

matrix protein (17). One clone, hlim4, contained a signal peptide and an 18 GLX repeat,

a representative feature of cement substances. A cement protein 36 (RIM36) of the tick

R. appendiculatus is regarded as an immunodominant molecule and a promising target

of eliciting antibodies in vaccinated cattle (17). The cement components are initially

secreted to assist in the attachment to the host tissue. The process by which cement

hardens are currently unknown and the individual protein comprising tick cement are

poorly characterized (17). Recently, transcriptome of the salivary glands of D. andrsoni

was analyzed by random sequencing of 1440 cDNA clones from the salivary glands of

adult female ticks collected during the early stages of feeding (18-24 hr) (2).

Interestingly, among all the categories, some of the highest percentage of potentially

secreted proteins was observed for the glycine-rich genes (67.7%). The relatively

abundance of messages potentially encoding cement-like protein in D. andersoni

salivary glands is consistent with the amount of attachment cement usually secreted by

ixodid ticks. Ticks from this group, which include Haemaphysalis, Dermacentor,

Rhipicephalus and Boophilus ticks, penetrate only the superficial epidermis of the host’s

skin and mechanical support is entirely dependent on the cement secreted (2).

62

With regard to vaccine targeting, no consensus agreement is obtained as to

whether an exposed or a concealed antigen is the best choice (67, 76, 140). The use of a

concealed antigen, as the commercial vaccines in cattle, can successfully induce

protective immunity against tick feeding. However, immunity to concealed antigens

may not prevent the attachment and feeding of the ticks or the transmission of tick-

borne infections. In addition, immunity from this vaccine is short-lived because natural

infestation does not stimulate a response to the concealed antigen (35, 138). Salivary

gland proteins are exposed antigens that are injected into the host during blood feeding.

Resistance to salivary gland proteins is naturally acquired by multiple tick infestation or

immunization with crude tick extracts (128, 132). The positive aspects of salivary gland

antigens as potential candidates for the regulation of tick attachment and feeding (92)

and transmission blocking vaccines (27, 84, 131) are superior beyond the drawbacks of

the reduction of their antigenicity under the pressure of host immunity (111) or their

inferior immunity compared to that induced by concealed antigens (133). In particular,

the immune response induced by the exposed antigen is expected to be continually

boosted by the natural exposure of ticks to livestock in the field.

The salivary glands of ticks produce a cocktail of molecules having different

functions (93-95, 129). One of the categories is the cement components that are

initially secreted from the salivary glands and enable the tick to attach to the host.

Several studies have shown the attractiveness of the cement protein as vaccine

candidates. Vaccination with the recombinant p29, the glycine-rich extracellular matrix

protein from H. longicornis, led to a significant reduction of the engorged body weight

in adult ticks and the mortality of both larvae and nymphs (75). Another candidate,

HL34, could induce morbidity and mortality in adult and nymphal ticks of the same tick

63

species (115). The recombinant cement cone protein RIM36 has been characterized

from R. appendiculatus, in which it was indicated that this cement component could

potentially elicit strong antibody responses in cattle exposed to feeding ticks (17).

When a putative tick cement protein (64P) from R. appendiculatus was examined as a

vaccine target, a cross-reactivity of anti-sera raised against several truncated forms of

recombinant 64P with several tissue extracts, suggesting that this cement protein was a

promising candidate targeting both exposed and concealed antigens (112). Moreover, it

was shown that vaccination with the recombinant 64P cement protein induced

protective humoral and cell-mediated immune responses in addition to antigenic cross-

reactivity with the other tick species, suggesting its potential for use as a broad-

spectrum anti-tick vaccine (113).

In the present study, I identified immunodominant antigens from the salivary

glands of H. longicornis and several novel putative cement proteins were obtained.

Further study on the recombinant expressed individual protein will clarify the role of

these proteins for tick attachment and their possible use in a recombinant cocktail

vaccine.

3-5. Summary

The components of tick saliva have many effects that may aid the vector during

engorgement and could be important in evading host responses. Animals acquire

resistant to tick bites, a phenomenon known as tick immunity is partially mediated by

antibody. The present study showed the results of immunoscreening of cDNA

expression library from salivary glands of H. longicornis using tick sensitized rabbit

serum. Screening of 500,000 library clones yielded 17 positive clones. As a result of

64

BLASTP search, 8 genes gave best match to hypothetical protein from H. longicornis

and one clone matched with HL35 antigen U from the same tick species. Eight novel

proteins possess common characteristic of adhesive cement substance were obtained.

Expression of these genes was confirmed by RT-PCR. From this study, it is of great

interest in further evaluating the vaccine potency of these immunodominant antigens.

65

Table 4. Categorization of the positive clones from immunoscreening. Sequence

Id Accession

number Genbank match* Species of NR

match

E-value Signal

peptide

hlim1 AB259293 No matches found - - T hlim2 AB259292 No matches found - - T hlim3 AB252633 >gi|12060354|dbj|BAB20558.1|

HL35 antigen U

H. longicornis 1e-119 S

hlim4 AB259294 No matches found - - S hlim6 - >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 S

hlim7 - >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 S

hlim8 - >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 S

hlim9 - >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 S

hlim10 AB259299 No matches found - - T hlim11 - No matches found - - T hlim12 - >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 T

hlim13 AB259296 No matches found - - S hlim14 AB259298 >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 S

hlim17 - >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 S

hlim18 - >gi|68051227|dbj|BAE02556.1|

hypothetical protein

H. longicornis 0.055 S

hlim20 AB259297 No matches found - - T hlim21 AB259295 No matches found - - T

*BLASTP performed with the Blosum62 matrix against the non-redundant (NR) protein database of NCBI; S, a secretory signal sequence was found; T, truncated sequence. Note; hlim10 and hlim11 are identical sequences (AB259299), 8 hypothetical proteins are the same sequences (AB259298)

66

Fig. 14. Multiple amino acid sequence alignment of novel proteins. The CLUSTAL

alignments of 8 novel proteins obtained by immunoscreening are shown (hlim1, hlimi2,

hlim4, hlim10, hlim13, hlim20 and hlim21; accession number AB2529293,

AB2529292, AB2529294, AB2529299, AB2529296, AB2529297 and AB2529295,

respectively). The unique features of cement protein, GLX repeats, are highlighted by

boxes. Residues conserved in all sequences are marked with black shadow. Similarity

is marked with a dark grey and light grey shadow. Note; hlim10 and hlim11 are

identical sequences (AB2529299).

67

Fig. 15. Multiple sequence alignments of the HL35 homologues. Amino acid sequence

alignments of hlim3 (AB252633), previously reported HL35 (BAB20588), and its

homologue, HL34 (BAB1376), are shown. The GLX repeats are underlined. Residues

conserved in all sequences are marked with black shadow. Similarity is marked with a

dark grey shadow.

68

Fig. 16. Analysis of mRNA encoding for immunodominant antigens in salivary glands

of unfed and partially fed female H. longicornis. Total RNA was isolated from the

salivary glands of unfed and partially fed female ticks. The RT-PCR reaction with

specific primers of each gene was performed. Primers specific for the H. longicornis

actin gene were used as an internal control for the RT-PCR reaction.

Unfed fed

hlim3

hlim1

hlim2

hlactin

hlim13

hlim14

hlim20

hlim21

hlim4

69

Chapter 4

Characterization of Haemaphysalis longicornis recombinant cement-

like antigens and evaluation of their vaccination effects

4-1. Introduction

Ticks are ectoparasites found in almost all parts of the world and surpass all

other arthropods in the number of diseases they transmit to animals and humans. Tick-

borne diseases in domestic animals are major constraints in livestock production,

especially in developing countries. Although tick control strategies rely extensively on

the use of chemical acaricides, this type of control is becoming less sustainable due to

the development of resistance against acaricides among ticks and the undesirable

contamination of the environment and animal food products (137). The limitation in the

current control measures against ticks and tick borne diseases have stimulated research

in alternative method. Anti-tick vaccination may be an ideal, affordable, and

sustainable way of controlling tick-borne diseases. There is currently only one

commercially available tick vaccine against R. (B.) microplus (54, 135). This midgut

membrane-bound protein antigen (BM86) can successfully induce protective immunity

against tick feeding. Antibodies bind to epitopes on the midgut cells of the feeding tick

causing damage and leakage of blood into the body cavity, killing the tick or reducing

fecundity. Concealed antigens are those found on the tick gut wall and normally not

presented to the host. Therefore, immunity to concealed antigens may not prevent the

attachment of the ticks or the transmission of tick-borne infections. In addition,

immunity from this vaccine is short-lived because natural infestation does not stimulate

a response to the concealed antigen (35, 138).

70

Salivary gland proteins are exposed antigens that are injected into the host

during blood feeding. Resistance to salivary gland proteins is naturally acquired by

multiple tick infestation or immunization with crude tick extracts (128, 132). As such,

salivary gland antigens are potential candidates for a transmission blocking vaccine (84,

131) and the regulation of tick attachment and feeding (92). Immune responses induced

by exposed antigens are expected to be continually boosted by natural exposure of

livestock to ticks in the field. Several exposed antigens have been identified, expressed

as recombinant proteins, and evaluated as vaccine candidates (75, 112, 113, 115). One

of such promising target molecule is the cement protein (112, 113). The cement

components are initially secreted to assist in the attachment to the host tissue. A cement

protein, RIM36, of the tick R. appendiculatus, is regarded as a strong antigenic

molecule target for eliciting antibodies in cattle (17). Furthermore, a putative tick

cement protein (64P) from R. appendiculatus is another vaccine target of interest (112).

A cross-reactivity study of anti-sera raised against several truncated forms of

recombinant 64P with several tissue extracts suggested that this cement protein may be

a promising candidate targeting both exposed and concealed antigens for use as a broad-

spectrum anti-tick vaccine (113).

From chapter 3, I identified immunodominant antigens from the salivary

glands of H. longicornis by immunoscreening. Several sequences that code for putative

cement proteins were obtained. Further studies of recombinant expressed individual

proteins were performed to clarify the role of these proteins for tick attachment and

their possible use as a recombinant cocktail vaccine. In the present study, 2 genes

encoding cement-like antigens, hlim2 and hlim3, obtained from the salivary gland

cDNA library of H. longicornis, were selected for expression as recombinant proteins.

71

Their potential as anti-tick vaccine candidates is discussed herein.

4-2. Materials And Methods

Ticks

The parthenogenetic Okayama strain of H. longicornis has been maintained by

feeding on rabbits and mice for several generations in our laboratory since 1997. The

adults and nymphs were used in this experiment (36).

Animals

All animal experiments were conducted in accordance with the Standards

Relating to the Care and Management of Experimental Animals promulgated by

Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan

(Allowance No. 17-77).

Nucleotide accession numbers

The nucleotide sequences reported in this thesis will appear in the

DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers

AB252633 and AB259292.

Analysis of hlim2 and hlim3 mRNA expression in different tick tissues

Total RNA was prepared from salivary glands, midguts, ovaries, synganglions,

and carcasses (remnants of ticks after the removal of salivary glands, midguts, ovaries,

and synganglions) of partially fed ticks using the TRI® reagent, as previously described

in chapter 1. RT-PCR was performed using the Takara one-step RNA PCR kit (Takara,

72

Tokyo, Japan) according to the manufacturer’s instructions. Primers specific for the H.

longicornis actin gene were used as an internal control for the RT-PCR reaction. A

negative-control reaction was performed without adding reverse-transcriptase to

exclude some contamination of genomic DNA.

Subcloning of hlim2 and hlim3 cDNA sequences into the pGEX-4T-3 expression

vector

The PCR amplification of the pBluescript phagemid harboring the hlim2 and

hlim3 cDNA sequence for expression was performed using primers flanking the coding

sequences of each gene. A truncated hlim2 was amplified using the hlim2 forward

primer fused in-frame with the GST gene (5’AGGAATTCGGCACGAGGGGGA3’) and

the hlim2 reverse primer (5’GCGAATTCTTATGACCGTGCA3’). The full-length

hlim3 was amplified using the hlim3 forward primer

(5’GCGAATTCTCATGACGCCTGT3’) and the hlim3 reverse primer

(5’GCGAATTCCATCATGAAGATA3’). The start codon (ATG) and stop codon (TTA,

TCA) are indicated in bold. The EcoRI restriction sites are indicated in italics. The

PCR reaction conditions were as follows: 94°C for 10 min, 30 cycles of 94°C for 1 min,

50°C for 1 min, 72°C for 2 min, and 1 cycle of 72°C for 7 min. The expression vector

pGEX-4T-3 (Amersham Pharmacia Biotech, Tokyo, Japan) was digested with EcoRI,

treated with calf intestinal alkaline phosphatase (CIAP), and purified using the

Geneclean II kit (Q-BIO gene®, Bio101 system, MP Biomedicals, CA, USA). The

insert DNA was digested with EcoRI, purified using Geneclean, and ligated into the

compatible ends of pGEX-4T-3 using the DNA ligation kit version 2.0 (Takara, Tokyo,

Japan) according to the manufacturer’s instructions. The ligation product was

73

transformed into E. coli DH5∝. Transformants grown on the plate were selected for

plasmid DNA mini-preparation using alkaline lysis methods. After restriction digestion

analysis, positive clones with correct orientation were selected for sequencing. The

recombinant constructs were transformed into E. coli BL21 cell for expression.

Expression and purification of the GST-hlim2 fusion protein

A single colony of E. coli BL21 haboring the recombinant pGEX-hlim2 plasmid

was grown overnight in 5 ml LB-ampicillin broth. The culture was diluted 1:50 in 200

ml of a fresh LB-ampicillin medium and grown until O.D600 reached 0.4. Then,

isopropyl-beta-D-thiogalactopyranoside (IPTG) induction was performed with a final

concentration of 0.5 mM at 25°C overnight. The cells were pelleted by centrifugation at

4,000 ×g for 20 min at 4°C. The pellet was resuspended in a 10 ml TNE buffer (50

mM Tris-HCl pH 7.5, 2 mM EDTA, 0.1 M NaCl) and further incubated at room

temperature for 10 min in the presence of a 20 μg/ml final concentration of lysozyme.

The cell suspension was lysed by sonication 3 times, 2 min each time, and incubated on

ice for 1 hr with 1% Triton-X 100 in PBS. After repeat sonications, the suspension was

centrifuged at 15,000 ×g for 20 min at 4°C. The supernatant containing soluble GST-

hlim2 was collected for further purification by glutathione sepharose 4B according to

the manufacturer’s instructions (Amersham Pharmacia Biotech, Tokyo, Japan).

Expression of GST-hlim3 fusion protein

The expression of E. coli BL21 harboring the recombinant pGEX-hlim3 plasmid

was performed in a 1 L culture at 37°C for 4 hrs with a 1 mM final concentration of

74

IPTG. After the cells were lysed by sonication, the suspension was centrifuged at

15,000 ×g for 10 min at 4°C, and the supernatant was discarded. The pellet was

washed 3 times by sonication in 1% triton-X-PBS and one time in PBS. The pellet was

resuspended with 10 ml of 8 M urea in a urea-free buffer (50 mM Tris-HCl pH 8.0, 1

mM DTT, 1 mM EDTA). The mixture was incubated at room temperature for 1 hr and

centrifuged at 15,000 ×g for 20 min at 4°C. The supernatant was transferred to a

dialysis tube, and slow removal of urea was performed by stepwise dialysis against 1 L

of a 4 M and 2 M urea solution, one hour each, and with a urea-free buffer 3 times, 30

min each time. The supernatant was further dialyzed against 2 L of a urea-free buffer

overnight at 4°C. After centrifugation at 15,000 ×g for 20 min at 4°C, the supernatant

was collected. Determination of the fusion protein concentration was performed by

SDS-PAGE in comparison to the known concentration of the standard BSA protein.

Immunization of mice using GST-hlim2 and the GST-hlim3 fusion protein

Nine ddy mice (female, 6-weeks old) were divided into 3 groups for the

immunization and challenge infestation experiment. The animals were immunized for

totally 4 times with the control GST protein, GST-hlim2, and the GST-hlim3 fusion

protein. First, immunization was performed by injecting 100 μg of the protein mixed

with equal volume of Freund’s complete adjuvant intraperitoneally. The booster was

given at 2-week intervals with the same amount of protein mixed with Freund’s

incomplete adjuvant. Blood samples were collected from the mice tail vein after the

third booster. The host immune response to the immunization was analyzed by Western

blot analysis of immunized mouse sera with the immunoblots of recombinant proteins.

75

Immunization of rabbits using GST-hlim2 and the GST-hlim3 fusion protein

Six Japanese white rabbits were immunized for totally 4 times with the control

GST protein, GST-hlim2, and the GST-hlim3 fusion protein. First, immunization was

performed by subcutaneous injection of 500 μg of the protein mixed with Freund’s

complete adjuvant. The booster was given at 2-week intervals with the same amount of

protein mixed with Freund’s incomplete adjuvant. Blood samples were collected from

the marginal ear vein after the third booster.

Analysis of native hlim2 and hlim3 in salivary glands by Western blotting

Salivary glands of 4-day fed H. longicornis were dissected, washed with ice-

cold PBS, and kept at -80°C until used. The tissue was homogenized using a tissue

homogenizer, sonicated for 15 s 3 times, and centrifuged at 15,000 ×g for 20 min at

4°C. The supernatant was collected, and the protein concentration was measured using

a BCA kit (Pierce, Rockford, IL, USA). Salivary gland extract was used as an antigen

for Western blotting by electrophoresing on a 12% polyacrylamide gel. The protein was

transferred onto a polyvinylidine difluoride (PVDF) membrane. The membrane was

incubated in 3% skim milk in PBS for 1 hr at room temperature and further incubated

with mouse sera (1:100) and goat anti-mouse-conjugated horseradish peroxidase (Dako

Cytomation, Tokyo, Japan, 1:2,000). The signal was detected by 3,3-diaminobenzidine

tetrahydrochloride (DAB) staining.

Challenge infestation

76

The antibody titer of animal immunized with GST-hlim2 and GST-hlim3 was

evaluated using ELISA and Western blot, respectively. When the antibody titer reached

1:5,000 to 1:8,000 dilution, the animal were challenged with ticks. Three ddy mice per

group were used for the challenge infestation. Ten days after the third booster, 12 H.

longicornis nymphs per mouse were applied to the backs of the mice with the help of a

polypropylene cap fixed with cement mass as described elsewhere (59). Sixty unfed

adult ticks were applied on shaved ears of immunized rabbits as described elsewhere

(36). The vaccine effect was determined 24 hrs later by visual examination of the

attachment rate (number of tick attach on the host) and duration of feeding (the time

since attachment was observed until engorgement was completed). The engorged body

weights, molting rate, egg weight, and mortality rates were determined post-

engorgement.

Statistical analysis

All data are presented as a mean ± standard error or percentages where

applicable. Differences are considered to be statistically significant if the P value was

less than 0.05 in the Student’s t test.

4-3. Results

Sequence analysis of hlim2 and hlim3

The nucleotide sequences and deduced amino acid sequences of hlim2 and hlim3

are shown in Fig. 17. The hlim2-truncated sequence (Fig. 17 a) (accession number

AB259292) is 696 bp in length, with an open reading frame (ORF) of 539 bp. The

deduced amino acid is 179 aa with an expected molecular mass of 16.5 KDa and a pI of

77

8.85. This protein has no similarity with any sequence reported in the database. The

hlim2 protein has high glycine (34.6%) and serine (14.53%) contents. Several glycine-

rich repeats (GGXG, GXGG) were observed, as reported for the R. appendiculatus

glycine-rich protein (67). The presence of GLX repeats, the common features of

cement component were shown.

The hlim3 full-length sequence (Fig. 17 b) (accession number AB252633) is

1,089 bp, has an ORF extending from position 40 to position 1,003, and codes 321

amino acid residues with a predicted molecular mass of about 33 kDa and a pI of 9.1.

This deduced amino acid has a secretory signal peptide signature. The hlim3 sequence

shared 41% and 83% identities with previously reported H. longicornis salivary gland

proteins, HL34 and HL35, respectively (115). The common features of the adhesive

protein, YPG motifs, within the tyrosine-rich domain or PXP motifs in the c-terminal

proline-rich domain were observed as described by Tsuda et al (115).

The expression of hlim2 and hlim3 mRNA transcripts in different tick tissues

The expression of the mRNA transcripts of hlim2 and hlim3 was studied using

RT-PCR (Fig. 18). Both mRNA transcripts were expressed predominantly in salivary

glands. The hlim2 transcript was expressed in every organ except the midgut. For hlim3,

the mRNA expression was observed in the salivary gland, synganglion, and carcass. The

RT-PCR internal control was performed using the actin gene.

Expression and purification of recombinant hlim2 and hlim3

The heterologous expression of recombinant hlim2 in E. coli was performed.

78

The GST fusion protein with an expected size of 42 kDa was obtained (Fig. 19 a). The

recombinant hlim3 was expressed with the expected size of 59 kDa. Although the

expression conditions were optimized with various concentrations of IPTG at different

temperatures, the protein did not become soluble (data not shown). For this reason,

hlim3 was expressed as the inclusion body, solubilized using urea, and refolded by

stepwise dialysis as described in materials and methods (Fig. 19 b). The purification of

the refolded hlim3 gave a low yield (less than 300 μg from 1 L culture). Therefore, the

hlim3 inclusion body was used for immunization.

Analysis of native hlim2 and hlim3 in salivary glands by Western blotting

The Western blotting of a salivary gland extract and mouse anti-hlim2 and anti-

hlim3 is shown in Fig. 20. The mouse anti-hlim2 reacted with 64 kDa and 26 kDa

proteins, whereas anti-hlim3 reacted with a 33 kDa protein from a salivary gland extract.

The reaction with the 26 kDa protein possibly resulted from a cross-reaction between

the anti-GST antibody and the GST protein in the salivary glands of ticks (50, 112, 113)

It is not known whether the molecular mass of the native hlim2 is 64 kDa, since the full-

length sequence of this clone is not yet available.

Challenge infestation in mice

The challenge infestation was performed as described in materials and methods.

The attachment rate of ticks fed on GST-hlim3-immunized mice (34.2 ± 17.6%) was

significantly lower (p < 0.05) than that in the control group immunized with GST

(100%) after the first day of tick application (Fig. 21). The feeding duration was

79

prolonged, as indicated by the significantly lower engorgement rate (p < 0.05) on day 3

(4.8 ± 3.9%) and day 4 (23.8 ± 3.8%, p < 0.01) of feeding (Fig. 22). Most of ticks

(95%) were engorged after 6 days of feeding. In contrast, GST-hlim2-immunized mice

exhibited no apparent differences in attachment and feeding periods from those

observed in the control group. However, the body weights of engorged ticks fed on

GST-hlim2-vaccinated mice (3.93 ± 3.9 mg) were significantly lower (p < 0.01) than

that of the control group (4.37 ± 0.05 mg) as shown in Fig. 23. The vaccination was

found to have no effect on tick molting, since all ticks were able to progress to the adult

stage.

Challenge infestation in rabbits

Challenge infestation in rabbits immunized with recombinant proteins was

performed using adult female ticks. There were no significant difference in the

attachment rate or feeding period of ticks fed on GST-hlim2 or GST-hlim3 immunized

rabbits in comparison to the control group. However, significant reduction of engorged

body weight of tick following feeding on GST-hlim2 (147 ± 45 mg, p < 0.01) and GST-

hlim3 (163 ± 37 mg, p < 0.05) in comparison to the control GST immunized group (184

± 32 mg) was observed (Fig. 24). An apparent reduction in egg weight was observed

for adult ticks fed on GST-hlim2 immunized rabbit (120 ± 13 mg, p < 0.05) compared

to the control value (153 ± 15 mg) (Fig. 25). However, this affect cannot be observed in

tick fed on GST-hlim3 immunized rabbit (150 ± 29 mg).

4-4. Discussion

Several studies have demonstrated that vaccination with defined protein

80

antigens is able to induce significant immunity to tick infestation (140). As a vaccine

target, salivary glands are a rich source of exposed antigens that can stimulate the host

immune response following natural infestation. As reported in chapter 3, I identified 17

genes encoding immunodominant antigens from the salivary glands of H. longicornis.

These genes were obtained by immunoscreening of a salivary gland cDNA library using

a tick-sensitized rabbit serum. Of the 17 positive clones obtained, 8 sequences did not

yield any similarities with known proteins, in addition that one sequence shared

similarity with the HL35 antigen U. These 9 sequences were found to possess

characteristics in common with cement proteins that may assist ticks in their attachment

to the host during blood feeding. To test whether or not this immune response was

protective, individual genes were selected for expression as recombinant proteins and

the vaccine potency was evaluated. In this study, 2 genes that gave a strong positive

reaction with a rabbit serum, hlim2 and hlim3, were selected for further study. The

hlim2 mRNA transcript was equally expressed in the salivary gland of unfed and fed

ticks, whereas hlim3 was upregulated during blood feeding. The analysis of mRNA

expression in various tick organs suggested that both genes were also expressed in

tissues other than the salivary glands. These 2 genes were subcloned and expressed as a

recombinant GST fusion protein using a bacterial expression system. The recombinant

GST-hlim2 was expressed as a soluble protein at 25°C, whereas GST-hlim3 was

expressed as an inclusion body. The recombinant GST-hlim2 and GST-hlim3 were used

for the immunization of mice. Challenge infestation was performed using H.

longicornis nymphs and adult ticks fed on vaccinated mice and rabbits, respectively. In

comparison to control GST, the ticks that fed on GST-hlim2-immunized mice had

significantly lower engorged body weight, although the attachment and engorgement

81

process was unaltered. On the other hand, ticks fed on mice vaccinated with GST-hlim3

showed a significantly lower attachment rate 24 h post-infestation, which prolonged the

engorgement rate on day 3 and day 4. However, those ticks were later attached and fed

until repletion with no significant difference in their engorged body weight.

Vaccination of rabbits using GST-hlim2 and GST-hlim3 separately could reduce

engorged body weight of adult ticks. However, apparent difference in oviposition can

be observed only in ticks fed on GST-hlim2 immunized rabbits. These results suggested

that antibodies against immunodominant cement-like antigens in vaccinated animals

could interfere with tick feeding in some way.

Both hlim2 and hlim3 proteins contain glycine repeats and a glycine-rich

region. The glycine-rich proteins have been found to be expressed in the salivary

glands of R. appendiculatus (17, 81, 112) and A. variegatum (80) and are believed to be

a component of tick cement, the proteins of which are rich in glycine, serine, leucine,

tyrosine, and proline (65). A tick attachment site usually consists of a cement cone that

protects the tick hypostome during feeding and secures the site of skin penetration (65).

Since cement proteins are essential for tick attachment and feeding on the host, there is

interest in using cement proteins or their derivative as anti-tick vaccine components.

Immunization of rabbits with purified cement proteins elicit delayed-type

hypersensitivity to the antigen (107). The hypersensitive rabbit demonstrated resistance

to feeding of the R. appendiculatus tick but a slightly enhanced feeding of

Rhiphicephalus pulchellus ticks. Vaccination with recombinant p29, glycine-rich

extracellular matrix protein from H. longicornis led to a significant reduction of the

engorged body weight in adult ticks and the mortality of both larvae and nymphs (75).

HL34, another candidate, was reported to induce morbidity and mortality in adult and

82

nymphal stages of the same tick species (115). The recombinant hlim3 studied in this

work shared 41% amino acid identity with HL34. However, no mortality of ticks fed on

vaccinated mice was observed. Instead, this recombinant protein seemed to interfere

with tick attachment with an unknown mechanism. Visual observation indicated that

ticks fed on vaccinated animals accumulated on the polypropylene cap instead of

seeking a host.

A putative tick cement protein (64P) from R. appendiculatus is currently a

promising vaccine candidate that was used for vaccine targeting for both exposed and

concealed antigens (112). As reported with regard to the vaccination effects of cement

protein 64TRP, most adult R. sanguineus on vaccinated guinea pigs were unattached

and observed to be crawling on the retaining gauze as if attempting to leave the host 24

hr post-infestation (113) As discussed by those authors, if the ticks had not been

restrained within the feeding chambers, they would most likely have abandoned the

immunized animals and sought alternative hosts.

Cement is among the initial chemical compounds that ixodid ticks secreted and

inject into the feeding site after proboscis penetrate host skin (17). Cement is critical for

tick attachment and prevention of host immune response molecules for interacting with

proboscis. Since the 9 sequences including hlim2 and hlim3 identified in the present

study possessed common characteristic with cement protein, it is conceivable to

speculate the mice immunized with hlim3 mounted a strong immunity against cement

proteins that prevented tick attachment and subsequent feeding.

In the present study, the recombinant putative cement proteins from H.

longicornis were expressed, characterized, and preliminarily evaluated their vaccine

potency. The complementary effects of hlim2 on tick engorged body weight and hlim3

83

on tick attachment indicate the suitability of the 2 recombinant proteins for use in a

cocktail vaccine.

4-5. Summary

Several immunodominat antigens were identified from salivary gland cDNA

library of H. longicornis by immunoscreening. Two genes encoding cement-like

antigens were selected for expression as recombinant proteins in E. coli. Immunization

of animals with individual recombinant protein affected blood feeding and oviposition

of immature and mature ticks. Cement protein is one of the promising vaccine

candidates for several tick species. The result obtained from this study suggested the

possibility to use these recombinant proteins for future development of cocktail vaccine

against tick infestation.

1 GGCACGAGGGGGAGCTGGACTTGGAGCCCCTGGCGCTAGCTTGCCAGGAGCCTCCGGAAG

1 GGCACGAGGGGGAGCTGGACTTGGAGCCCCTGGCGCTAGCTTGCCAGGAGCCTCCGGAAG

a)

84

A R G G A G L G A P G A S L P G A S G S 20

61 CGGCAGCCCTGCTCTAAGCGGTCAACCAGGATTCGGCTTCGGAGGCTTCTCCGTCGCACC

G S P A L S G Q P G F G F G G F S V A P 40

121 CTATGGCGGTCAATGGGGTGTCCCGCAAGGCTTCTATGGAGTGTACCCAGGATTCAGCTA

Y G G Q W G V P Q G F Y G V Y P G F S Y 60

181 CGGATTCCCCGGTTACGGTAACGGTTATGATGCCTTCGGATTGGGTGGTGGATACGGCGG

G F P G Y G N G Y D A F G L G G G Y G G 80

241 CTACGGACCCTTCGGTGTCAACGGCTTCTACTCTCCACTCGCCGGCTACGGCTTCGGCGG

Y G P F G V N G F Y S P L A G Y G F G G 100

301 CTATGGCCTGGGCGGCTACCCTGCCCTCGGCGGCTACGGCTTCGGTGGCTATGGCTACCC

Y G L G G Y P A L G G Y G F G G Y G Y P 120

361 ATTCTTCGGTGGATTGGGAGGTGCTTACGGCTTTGGCCCCTCAGGTTTCTCCGGTAGCAG

F F G G L G G A Y G F G P S G F S G S S 140

421 CGCAAGCCAGAGTTCCCTGGGTGGTGCAGGTCTAGGTGCATCCGGCGCTTCTGGAGCGGC

A S Q S S L G G A G L G A S G A S G A A 160

481 AGCAGGCTCCAGCGCCAGGTCCGGATCCAGCTCCAGCAGCGGCGCCGCTGCACGGTCATA

A G S S A R S G S S S S S G A A A R S *

541 AGCGTAACTTCTGCCCTAACTAAGTTACTACCAACAAATTTCTTGACGGGCTTTCAAGGC

601 TGTTCTATATTTGGGCTGACCAACCTACAGGTTACAACGAGTCTAAAAACAATAAATTA

661 TGATTGTTCAATTTGAAAAAAAAAAAAAAAAAAAA

Fig. 17. Nucleotide sequences of genes encoding hlim2, hlim3 and their deduced amino

acid sequences.

a) Truncated nucleotide sequence and deduced amino acid sequence of hlim2. The stop

codon (TAA) is underlined with a thick line. Several glycine-rich repeats (GGXG,

GXGG), as reported for the R. appendiculatus glycine-rich protein motifs are

underlined. The GLX repeats, the common features of cement component are

highlighted by boxes.

85

1 GGCACGAGGCCGACCAGCATCACAGGAGAACGCGTAATCATGAAGATATCCGCTGCGCTC

M K I S A A L 7

61 ACACTTCTTTCCATGGTAGCCTATGCGGCAGGAGCCGTGAAGACCTTCTATAAAGCGGCA

T L L S M V A Y A A G A V K T F Y K A A 27

121 AAGCTCAAGAGCACTGTTGCTGCCGCTGTTCCCGTAGCTGTGTCACCCACTGAGGCTTTT

K L K S T V A A A V P V A V S P T E A F 47

181 CCTGAGCTCCTTGGTTCGACCTTGGTTAATGGTGGCACGGGTACGGTAGCAGTTTCGTCC

P E L L G S T L V N G G T G T V A V S S 67

241 ACCAGCAATCCTGTTGGTACCAGCGTCAGTCATGGAACCACTACTCACCAGGTCACTCGA

T S N P V G T S V S H G T T T H Q V T R 87

301 ACGAACATATATCCTGGCATCCCTGGCGATGCTTACTACAACCCCTATTTCCCAGCAGGC

T N I Y P G I P G D A Y Y N P Y F P A G 107

361 TATCCTGGATACGGTACAGGGCTGTACCCTGGCTTCCACAGCGGAGCTTATAACAGCTAC

Y P G Y G T G L Y P G F H S G A Y N S Y 127

421 CTCCGTTGGGCTTACCCTGGATTAAGTGCAGGTTTTTATCCCGGCTTCTTTCACTCAACT

L R W A Y P G L S A G F Y P G F F H S T 147

481 CGTCATGGATTCTCTAGCCCACTTTACCCTGGCCTTCTGGAAGCATCTCAGCACGGTCTT

R H G F S S P L Y P G L L E A S Q H G L 167

541 TTTGGAGGAGCCTACTCCAGATACCTGAGCGGAGTGTATCCTGGACTCTTCGGAGGGGAA

F G G A Y S R Y L S G V Y P G L F G G E 187

601 TACGGAAGTCTCTATGGCTCTCGCTTCTTCGGCAACGGTGCTTTGGAGAAGGTCCGAACG

Y G S L Y G S R F F G N G A L E K V R T 207

661 CATTTCGCAGGATACCAGAGAGAAGTTACTGTCACCGTCGACCCACTGACTGGGCTACCA

H F A G Y Q R E V T V T V D P L T G L P 227

721 GCGCCAGTTACGATTCCTGTTGCCAACCGCGTCGTGACGATCGAGCGCTTCGTACCATTC

A P V T I P V A N R V V T I E R F V P F 247

781 CCATCTCCGTATCCAGTACCGGTCCCAGCTCCTCACCCAGCCCCGTTCCCGTTGCCGCAC

P S P Y P V P V P A P H P A P F P L P H 267

841 CCTGGAGTCTCCAACCCAGTTTCGTCAACTACTCAAATGGGATCTACCACCACAGTAAGC

P G V S N P V S S T T Q M G S T T T V S 287

901 CAAGGTGGACTGGTCACTCCAGTCTCGGGTGGTGCAGATCTCGTTTCCGGCCAACAGGAA

Q G G L V T P V S G G A D L V S G Q Q E 307

961 GCTGTTGCGCAGACCCAGGCCCATGTTAATTTACAGGCGTCATGAAGACGGGTAATGTCA

A V A Q T Q A H V N L Q A S *

1021 CGCGACAAGCTTCGCAAAACAGTTAAATAAAATGGAATGTGAACAAAAATTAAAAAAAAA

1081 AAAAAAAAA

b)

86

b) Full-length nucleotide and deduced amino acid sequence of hlim3. The signal

peptides are underlined with a double line. The start (ATG) and stop (TGA) codons are

underlined with a thick line. The common features of the adhesive protein, YPG motifs,

within the tyrosine-rich domain or PXP motifs in the c-terminal proline-rich domain are

shown. The tyrosine-rich and proline-rich regions are underlined with a broken line. The

YPG motifs are indicated in bold, and the PXP motifs are in italics. The GLX repeats

within the deduced amino acid are highlighted by boxes.

87

Fig. 18. Analysis of hlim2 and hlim3 mRNA expression in different tick tissues.

The mRNA expression of hlim2 (a), hlim3 (b) and internal control actin (c) in different

tick organs was studied by RT-PCR. Lane 1, salivary glands. Lane 2, midgut. Lane 3,

ovary. Lane 4, synganglion. Lane 5, remnants of carcass after removal of the salivary

glands, midgut, ovary, and synganglion.

bp Mr 1 2 3 4 5

600 400

bp Mr 1 2 3 4 5

600 400

bp Mr 1 2 3 4 5

400 200

(a)

(c)

(b)

88

Fig. 19. The SDS-PAGE of purified recombinant GST/hlim2 and GST/hlim3. (a) Mr =

Low range molecular weight marker, hlim2 = purified GST/hlim2. (b) Mr = Low range

molecular weight marker, hlim3 = GST/hlim3 inclusion body.

kDa Mr hlim3

97

30

22

66

45

97

30

22

66

45

kDa Mr hlim2

a) b)

89

Fig. 20. Analysis of native hlim2 and hlim3 in salivary glands by Western blotting

Western blotting of salivary gland extracts from female ticks reacted with mouse sera.

Mr, pre-stained marker. Lane 1, anti-GST mouse serum. Lane 2, anti-hlim3 mouse

serum. Lane 3, anti-hlim2 mouse serum.

Mr 1 2 3

98

22

16

64

50

36

kDa

90

0

20

40

60

80

100

120

day0 day1 day2 day3

att

ach

men

t ra

te (

%)

GST

hlim2

hlim3

Fig 21. Comparison of attachment rate after application of H. longicornis nymphs on

GST, GST-hlim2 and GST-hlim3 immunized mice. Mice were immunized with GST-

hlim2, GST-hlim3 or GST alone, infested with H. longicornis nymphs as described in

materials and methods and the nymphal attachment rate were determined. Data are

presented as mean ± SE (* indicates p < 0.05).

*

91

0

20

40

60

80

100

120

day3 day4 day5 day6

en

go

rgem

en

t ra

te (

%)

GST

hlim2

hlim3

Fig. 22. Comparison of engorgement rate of H. longicornis nymphs fed on GST, GST-

hlim2 and GST-hlim3 immunized mice. Mice were immunized with GST-hlim2, GST-

hlim3 or GST alone, infested with H. longicornis nymphs as described in materials and

methods and the nymphal engorgement rate were determined. Data are presented as a

mean ± SE (* indicates p < 0.05; ** indicates p < 0.01).

**

*

92

3.50

3.70

3.90

4.10

4.30

4.50

4.70

GST hlim2 hlim3

engo

rged

body

weig

ht

(mg) GST

hlim2

hlim3

Fig. 23. Comparison of engorged body weights of nymphal ticks fed on GST, GST-

hlim2 and GST-hlim3 immunized mice. Mice were immunized with GST-hlim2, GST-

hlim3 or GST alone, infested with H. longicornis nymphs as described in materials and

methods and the nymphal engorged body weights was determined (** indicates p <

0.01).

**

93

140

145

150

155

160

165

170

175

180

185

190

GST hlim2 hlim3

engo

rged

body

weig

ht

(mg)

GST

hlim2

hlim3

Fig. 24 Comparison of engorged body weights of adult ticks fed on GST, GST-hlim2

and GST-hlim3 immunized rabbits. Rabbit were immunized with GST-hlim2, GST-

hlim3 or GST alone, infested with H. longicornis adults as described in materials and

methods and the engorged body weights was determined (* indicates p < 0.05, **

indicates p < 0.01).

**

*

94

^

100

110

120

130

140

150

160

170

GST hlim2 hlim3

egg

weig

ht

(mg)

GST

hlim2

hlim3

Fig. 25 Comparison of egg weights of adult ticks 3 weeks after drop-off from GST,

GST-hlim2 and GST-hlim3 immunized rabbits. Rabbit were immunized with GST-

hlim2, GST-hlim3 or GST alone, infested with H. longicornis adults as described in

materials and methods and the egg weights was determined. (* indicates p < 0.05).

*

95

General discussion

Control of ticks and tick-borne diseases of livestock depends almost exclusively

on the use of acaricides (41, 89). Widespread occurrence of tick resistance to acaricides

and environmental concerns prompted research into alternative methods for controlling

ticks (41, 140). Vaccines designed to reduce tick infestation, which are deployed as part

of integrated control measures involving reduced acaricide usage, are therefore being

actively explored. Development of new tick control measures requires the identification

of candidate genes that affect immune evasion and pathogen transmission. Functional

genomics has proved a valuable tool that can accomplish this task.

Successful tick feeding and pathogen transmission are both facilitated by

pharmacologically active molecules secreted in tick saliva (21, 121). Tick salivary

gland molecules inhibit host hemostasis, reduce pain and itch responses, and modulate

host innate and specific acquired immune defenses (43, 73, 94, 103, 131). Some

components of tick saliva are highly immunogenic and vaccination with salivary gland

proteins can interfere with the tick life cycle (75, 139). The composition of tick saliva

is interesting in the study of biology of host-parasite relationship, discovery of

biologically active components and identification of novel vaccine targets. For this

reason, the cDNA library from salivary glands of H. longicornis was constructed in this

study. Besides providing basic information on the genes expressed in tick salivary

glands, I have interested in defining genes encoding components that are secreted in

saliva as these play a critical role in the biology of tick feeding (102) and pathogen

transmission (84, 131). Since the function of many sequences described still unknown,

cloning and expression of selected cDNA sequences would give a better understanding

96

of tick blood feeding mechanism. RNA interference has emerged as one of the most

promising technologies for rapid systematic functional genomic analyses. The

application of RNAi to ticks has opened the possibility of manipulating gene expression

in these organisms (25, 26, 49). RNAi has been successfully used for the

characterization of tick genes and for the screening of tick-protective antigens (24). The

application of this technology to ticks could impact on tick vaccine development

through the identification and characterization of tick-protective antigens.

As an antihemostatic molecule, tick metalloprotease(s) appears to cleave

fibrinogen and fibrin, therefore inhibiting clot formation (32, 33). Metalloprotease also

may affect endothelial cell function and angiogenesis (33). Based on the information

obtained from the random sequencing of salivary gland cDNA library of H. longicornis,

4 genes encoding metalloproteases were selected for further study. Full-length cDNA

from 2 out of the 4 genes were obtained. At the mean time, another cDNA library was

constructed using vector-capping method, which has several advantages compared to

classical lambda Zap library. By using this method, approximately 95% of cDNA

clones are full-length. The EST database was constructed by random sequencing and

annotating of 10,000 cDNA sequences. The other 2 full-length metalloproteases were

got from this EST library. Phylogenetic analysis and mRNA expression profiles during

blood feeding of each gene indicated an evolutionary distinct and high degree of

diversity of this protein. The presence of these diverse enzymes might contribute to the

greater functional complexity of this molecule in tick saliva to facilitate blood feeding.

Tick metalloproteases contain N-terminal prodomain, which, as in most zymogens or

proenzymes, is believed to maintain enzyme latency and to be proteolytically excised to

permit catalytic activity, a process termed “activation”. Since to my best knowledge

97

there is no report on the processing mechanism of tick metalloproteases, one of the goal

of this work was the expression of the proenzymes, subsequently, we can ascertain

whether the removal of its prodomain by a pro-protein convertase, as in the case of the

activation of some matrix proteases and the ADAMs (86) or by autolysis, as in the case

of snake venom metalloproteases (88). Several evidences from this study indicated that

proenzyme activation of this protein possibly occurs through furin type pro-protein

convertase dependent pathway. In order to confirm this finding, further experiments

have to be done such as site-directed mutagenesis to confirm the furin cleavage site or

baculovirus expression of the proenzme in the presence of furin inhibitor. It is also of

interest to identify furin or other enzymes responsible for activating proenzymes in tick

salivary gland. Accordingly, understanding the pathway for metalloprotease processing

may provide novel targets for the development of tick control since disruption of

proenzyme activation may inhibit the production of active enzyme, as a consequence,

interfering tick biological processes including blood feeding mechanism.

Skin lacerated by feeding ticks shows defective healing responses since these

ectoparasites are able to remain attached and fed on their host for a long period of time.

Therefore, knowledge of the host-parasite interface in terms of tissue repair can

contribute to understanding of the feeding process, transmission of infectious agents,

and potentially development of new vector-blocking vaccine candidates that could

interfere with tick attachment and at the same time prevent pathogen transmission (2).

Vaccines have been shown to be a feasible tick control method that offers a cost

effective, environmental safety alternative to chemical control. However, identification

of tick protective antigens remains the limiting step in vaccine development. Herein,

immunoscreening was used as a tool to identify 17 genes encoding immunodominant

98

salivary gland antigens that elicit antibodies in the hosts. Interestingly, several of these

proteins are rich in glycine, serine and leucine, which are prominent in the overall

amino acid content previously determined for the cement substance of the related ixodid

tick R. (B.) microplus (6). Cement is among the initial chemical compounds ixodid

ticks secreted and inject into feeding site after proboscis penetrated host skin (17) and

critical for tick attachment and prevention of host immune response molecules for

interacting with proboscis. The cement material is in contact with Langerhans cells in

the host’s skin; these dendritic cells present tick antigens to the host immune system (5,

83). Since cement proteins are essential for tick attachment and feeding on the host,

there is interest in using cement proteins or their derivative as anti-tick vaccine

components (75, 112, 113, 115, 146). Hence, recombinant expression and evaluation of

vaccine potency of these cement-like proteins was performed in this study. Initially, 2

sequences that gave strong positive reactions from immunoscreening, hlim2 and hlim3

were selected for further study. Immunization and challenge infestation of nymphal

ticks was performed in mice and that of adult ticks was performed in rabbit. The adult-

on mice model was not investigated here due to the limit number of adult tick that can

apply onto mice and difficulties in attachment of adult tick on small animal such as

mice. Immunization of GST-hlim2 affected immature and mature ticks to the same

degree as evidenced by significant reduction of engorged body weight of nymphs fed on

mice, adult fed on rabbit and also reduction of oviposition. On the other hand,

immunization of mice with GST-hlim3 showed significant lower attachment rate,

prolong feeding period but no affect on engorged body weight of nymphs. In contrast,

adult ticks fed on GST-hlim3 immunized rabbits had no apparent difference in

attachment rate but showed significant reduction in engorged body weight. Data from

99

several studies provide evidence which may support the hypothesis that immature and

mature ticks have different sensitivities to host acquired resistance against tick

molecules (63, 64, 75, 146). Finding from my finding are therefore in agreement with

their studies of different vaccine effects on nymphs and adult ticks fed on GST-hlim3

immunized animals.

Cellular immunity is an important mechanism of resistance to ticks.

Characterizing the inflammatory reactions at tick feeding sties, particularly cell

migration, will enhance understanding of the anti-tick effect of vaccines. Hosts that

develop resistance following tick infestations show a cutaneous basophil

hypersensitivity reaction, with skin infiltration by basophils in hyperplastic epidermis in

guinea pigs (4), and infiltration of neutrophils, macrophages, eosinophils and basophils

in rabbits and cattle (42, 123). From the recent study of truncated constructs of 64P, a

secreted cement protein from salivary glands of the tick R. appendiculatous suggested

that tick feeding on immunized animals stimulated local inflammatory immune

response (involving basophils, eosinophils, lymphocytes, mast cells, macrophages and

dendritic-like cells) that boosted the immune status of vaccinated animals (113).

Though the vaccination effects of cement-like antigens in my study was not conclusive,

protective response may be similar to 64P protein. Mechanism by which the

immunization affect ticks infestation may be further investigated by histo-pathological

examination at the site of infestation. The composition of the surrounding host skin

epidermal/dermal proteins would indicates a strategy whereby the tick avoids rejection

at the tick-skin attachment site by host immune defense mechanism.

Taken together, the results from this study suggested that animal immunized

with recombinant cement-like antigens could confer partial protection against ticks

100

feeding. This data, in consistent with several studies, have emphasized the fact that a

single tick molecule may not confer an inclusive protective immunity. An effective tick

vaccine will require a cocktail of target antigens each mediating a physiological

function either independently or synergistically (106). Several evidences suggested that

anti-tick immunity induced by a cocktail antigen vaccine is more effective compared to

a single antigen vaccine (98, 136). The results from my study reveal the possibility to

use cement-like antigens as a candidate for cocktail vaccine. It is of crucial interest to

evaluate the individual vaccine potency of the remaining positive clones obtained from

immunoscreening and later, in combination as a cement-based cocktail vaccine.

101

Conclusion

Since tick saliva contains a mixture of pharmacologically active compounds that

are essential for creating and maintaining the blood pool or the feeding lesion in host

skin. As a vaccine target, salivary glands would be a rich source of antigens.

Identification and characterization of tick salivary gland proteins is of considerably

interest since such information might contribute to the improvement of methods for the

control of tick and tick-born diseases. Therefore, a cDNA library from the salivary

glands of hard ticks, Haemaphysalis longicornis, was constructed in this study. Random

sequencing and immunoscreening of cDNA clones was used as a tool to recruit key

molecules that might be useful for the development of tick control strategies.

In chapter 1, a cDNA expression library of salivary glands from the adult female

H. longicornis tick was constructed and randomly sequenced. The aim of this study

was to identify salivary protein profiles of tick during blood feeding stage. Based on a

similarity search of deduced amino acid sequences using the BLASTP program, it was

found that 65 (41%) of 158 sequences are novel proteins. The remaining 93 (57%)

sequences matched sequences reported in the GenBank database. Of these 93 sequences,

25 matched those previously reported from ticks. These 93 cDNA clones were

classified according to their possible functions. The majority of the studied sequences

were housekeeping ones. Several bioactive molecules, which were expressed during

blood feeding, were found in this library; they included antihemostatic,

immunomodulatory, or cement formation proteins. Therefore, it is of great interest in

further characterization of the biological activities or evaluation of vaccine potency of

these proteins.

102

In chapter 2, among the bioactive molecules obtained in the cDNA library, 6

genes encoding metalloproteases were studied. Since this enzyme is capable of

hydrolyzing various components of the extracellular matrix and blood coagulation

related proteins, molecular cloning and characterization of these molecules was

performed in order to clarify their roles in tick feeding mechanism. Comparative

analyses have shown the evolutionary distinct and different mRNA expression patterns

of each gene during blood feeding. The proteins are synthesized in a proenzyme latent

form that requires activation for proteolytic activity. This proenzyme contains a signal

peptide, prodomain and a metalloprotease/cysteine-rich domain. The recombinant

Escherichia coli expression of one gene, hlESTMP1 was performed and used to obtain

anti-hlESTMP1 antibody. The immunoblot analysis and indirect fluorescent assay

suggested that this protein is mainly expressed in the cytoplasm of the salivary gland

cells and the mature form of 34 KDa was detectable. Since understanding the

proenzyme activation mechanisms would provide the necessary information for the

design of a selective inhibitor, the processing of the proenzyme of hlESTMP1 was

demonstrated using a baculovirus expression system. Accordingly, the signal peptide of

full-length hlESTMP1 was cleaved by signal peptidase and the proenzyme was further

processed into 2 smaller fragments. The sequences of the fragments suggested that the

enzyme activation mechanism occurs via pro-protein covertase-dependent pathway.

Thus, to identify a novel target for the development of a tick control approach, the

methods by disrupting the proenzyme activation mechanism to inhibit the production of

an active mature enzyme or by exploring metalloprotease regulators, such as inhibitors

or blockers of enzyme function, by vaccinating host animals with this bioactive

molecule, are considered.

103

In chapter 3, to identify the salivary gland antigens that elicit antibodies in the

host, immunoscreening of salivary gland cDNA library was performed using sera of

rabbit repeatedly infested with ticks. A BLASTP search suggested that from 17 positive

clones obtained, 8 sequences matched with that of hypothetical H. longicornis sequence

and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no

match to any sequence reported in the database. Interestingly, the deduced amino acid

sequences of these novel genes possess common characteristics with cement proteins,

which assist ticks in their attachment to the host during blood feeding. The expression

of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences

showed to be upregulated upon blood feeding. These immunodominant antigens are of

particular interest as candidates for future cement protein based-tick vaccine. For this

reason, 2 genes encoding cement-like antigens, hlim2 and hlim3, were selected for

further elucidation of vaccine potency. In chapter 4, the results were discussed. The

recombinant proteins were expressed in E. coli as the GST fusion protein and used for

immunization. It was observed that the attachment rate of nymphal ticks fed on mice

immunized with GST-hlim3 was significantly lower than that in the control group

during the initial days of feeding. However, immunization with GST-hlim3 did not

affect the engorgement rate of the adult ticks. In sharp contrast, GST-hlim2 did not

influence the attachment rate and feeding period of ticks but had a significant reduction

in the engorgement body weight. Vaccination of rabbits with GST-hlim2 resulted in

significant reduction of engorgement body weight and oviposition of adult ticks

whereas vaccination with GST-hlim3 affected only engorgement body weight. These

data highlight the suitability of the 2 recombinant cement-like proteins for use in a

cocktail vaccine. Based on several studies, anti-tick immunity induced by cocktail

104

vaccine is likely to be more effective than single vaccine antigen. For this reason,

further studies are required to evaluate the remaining novel cement-like antigens and

compare the efficacy of the single and cocktail antigens.

In conclusion, this study reports result from sequence analysis of salivary gland

cDNA library. Several bioactive molecules that possibly play important role in

countering host defense systems were identified. In this report, 2 unprecedented

molecules including metalloproteases and cement-like proteins were selected for further

studied. This is the first report to evaluate the expression profiles upon blood feeding,

expression of the recombinant protein and proenzyme activation mechanism of

metalloproteases from the salivary glands of ticks. Another molecule, cement-like

antigens is one of the interesting vaccine candidates. Vaccination of animal using the 2

recombinant cement-like antigens can affect the tick feeding. The information obtained

from this study would assist in the development of tick control strategy. Further studies

with reverse-genetic approaches will provide further insight into the functions and

significances of these molecules in tick biology and parasitology.

105

Acknowledgements

This research work was carried out at the National Research Center for

Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary

Medicine (Obihiro Univ. Agric. Vet. Med.). This study was supported by Bio-orientated

Technology Research Advancement Institution (BRAIN) and the 21th century COE

Program (A-1) Ministry of Education, Culture, Science and Technology, Japan.

I would like to acknowledge Dr. Naoyoshi Suzuki, a president of Obihiro Univ.

Agric. Vet. Med. for providing me an opportunity to carry out research in this university.

I would like to express my deep and sincere gratitude to my previous

supervisor, Prof. Kozo Fujisaki (Obihiro Univ. Agric. Vet. Med. and Kagoshima Univ.)

for his intellectual instruction, detailed and constructive comments, invaluable

suggestions, patiently revising this dissertation and tireless supports throughout this

work.

I am deeply grateful to my present supervisor, Prof. Hiroshi Suzuki (Obihiro

Univ. Agric. Vet. Med.) for his critical comments, suggestions and supporting me to

complete this thesis.

I wish to express my warm and sincere thanks to Prof. Makoto Sugiyama (Gifu

University), Prof. Tadashi Itagaki (Iwate University), Prof. Yoshikazu Hirota (Tokyo

University of Agriculture and Technology) and Prof. Shin-ichiro Kawazu (Obihiro Univ.

Agric. Vet. Med.) for constitutive and invaluable suggestions, and patiently revising this

dissertation.

I would like to express my special gratitude to carry out my research under

excellent supervision of Prof. Xuenan Xuan, Assoc. Prof. Yoshifumi Nishikawa at

106

NRCPD, Assoc. Prof. Tomohide Matsuo at Kyorin University School of Medicine and

Dr. Naotoshi Tsuji (Natl. Inst. Anim. Hlt. Q). I also would like to acknowledge Prof.

Ikuo Igarashi, director of NRCPD, Assoc. Prof. Noboru Inoue, Assoc. Prof. Naoaki

Yokoyama, Assoc. Prof. Makoto Igarashi, Prof. Hirotaka Kanuka and Dr. Shinya

Fukumoto for their meaningful and helpful suggestion.

I warmly thank all my colleagues in research unit of Molecular and Vector

Arthropodology in NRCPD for their hospitality, technical and mental supports. I owe

special thanks to Mr. Takeshi Sakaguchi, the previous undergraduate student who

greatly contributed to my work, Dr. Takeharu Miyoshi (Natl. Inst. Anim. Hlt. Q) for his

technical supports and Dr. Namangala Boniface at the University of Zambia for his

critical review for manuscripts preparation.

A special appreciation to all the scientific- and non-scientific staff, as well as

students at NRCPD for their kind co-operation and help in many ways.

Lastly, I owe my loving thanks to my husband, Dr. Noritaka Kuboki, for his

help in Japanese translation, documents preparation and mental supports. Without his

understanding and encouragement, it would have been impossible for me to complete

this work. Special thanks are due to my father, mother and family for their loving

supports.

107

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