Ticks are hematophagous arthropod ectoparasites that feed on their vertebrate hosts
for prolonged periods from days to several weeks. To successfully complete their
blood meal they developed strategies to secure attachment on their host's skin, to
ease blood flow and to evade their host's inflammatory and acquired immune
responses. Ticks are also vectors of a number of major human and animal diseases
and salivary gland secretions have been implicated in transmission and
establishment of such infections.
Amblyomma variegatum is a hard tick, found in tropical areas. Infestation with A
variegatum is associated with exacerbation (not transmission) of dermatophilosis, a
skin disease of cattle caused by the actinomycete Dermatophilus congolensis. This
observation suggests that A variegatum ticks secrete molecules that can modulate
the host immune response in such a way as to both prevent potentially harmful
responses against themselves and change the pattern of infection and disease caused
by unrelated pathogens.
The purpose of this study is to identify and characterise saliva and salivary gland
molecules that could contribute to the development of novel anti-tick vaccines or
have potential therapeutical value for medical application such as non-steroid anti-inflammatory drugs. To identify such molecules, three methodological approaches
have been used.
A proteomics approach was carried out to identify proteins contained in saliva and
salivary glands of partially fed A variegatum ticks. A combination of protein
separation on one- and two-dimensional SDS-PAGE and peptide mass fingerprint
analysis of selected proteins through MALDI-ToF/MS (Matrix-Assisted Laser
Desorption/Ionisation- Time of Flight/ Mass Spectrometry) technology was
undertaken. Resulting monoisotopic masses of 16 selected spots (9) and bands (7)
were compared with proteins of Swissprot database, and with the 6 frame-translated
of deposited EST of Amblyomma variegatum, Amblyomma americanum,
Rhipicephalus appendiculatus, Ixodes scapularis and Ixodes pacificus (TIGR)
databases. A 48 kDa band matched a cathepsin L like proteinase previously
identified in B microplus and H longicornis ticks, and a 45 kDa protein matched an
EST which was homologous to silk fibroin of Bombyx mori. A 86 kDa band and a
66 kDa spot matched myosin of mammalian origin, the other proteins matched EST
of the tick databases but the functions and identities of those matches remain to be
determined. However, comparative analysis of protein pattern of saliva and salivary
glands of unfed and partially fed female A variegatum confirmed that feeding
induced the expression of a large number of proteins.
A genomics approach was also undertaken to identify salivary gland genes, which
are specifically expressed during feeding of female A variegatum-.
A cDNA library of salivary glands of partially fed female A variegatum was
constructed in the bacteriophage A,TriplEx2. The titre of the library was 8x10
pfu/ml with 90% recombinants. Immunoscreening was carried out with a
hyperimmune antiserum obtained from a rabbit immunised with salivary gland
extracts of unfed female A variegatum and with antiserum prepared from a calf
repeatedly exposed to feeding A variegatum. While both antisera recognised saliva
and salivary gland protein extracts on Western blot analysis, they failed to detect
expressed recombinant proteins after many attempts of screening the cDNA library.
Sequence comparison with GenBank® non-redundant (NCBI) and with
Amblyomma variegatum Gene Index (TIGR) databases of 33 randomly selected
cDNA clones was carried out. The comparative analysis revealed that one clone
shared sequence identity with boophilin, a thrombin inhibitor of B microplus; and
another cDNA displayed 31% homology with TCI83 of AvGI, which shared itself
28% sequence identity with an immunomodulator of Dermacentor andersoni. Six
cDNA clones shared sequence homology with molecules which functions are
related to wound healing, anticoagulation and cement cone formation. While
several cDNA clones did not match any sequences of either database others
matched sequences of genes, which functions, have yet to be ascribed.
A Signal Sequence Trap (SST) technique, using alkaline phosphatase as reporter
gene and COS-7 cells as the host, was used to isolate genes encoding type I
membrane and secreted proteins from a cDNA library of salivary glands of partially
fed female A variegatum. A total of 8832 clones were screened and 13 positive
clones were isolated amongst them, 6 displayed a signal peptide at the N terminal
end of their deduced amino acid sequence. Sequence comparison with sequences of
the GenBank® nr (NCBI) and AvGI (TIGR) databases revealed that one clone
shared 99% identity with TC3, a glycine rich protein from the AvGI database that is
believed to be a cement cone protein. The full-length sequence of this clone,
FLAv41E was isolated from the >,TriplEx2 cDNA library of salivary glands of
partially fed female A variegatum. FLAv41E encoded a 1221 bp cDNA with a 1053
bp open reading frame. The deduced protein contained 351 aa with 20 aa predicted
signal sequence its theoretical molecular weight was 30.9 kDa with a pi of 8.1. Its
secondary structure revealed domain similarities with a 29 kDa cement protein
previously characterised in salivary gland of Haemaphysalis longicornis ticks.
Reverse transcription PCR (polymerase chain reaction) amplification of FLAv4IE
gene revealed that it was expressed in the salivary glands of female A variegatum
from day 3 to 10 of feeding. A his-tagged FLAv41E protein minus its signal
peptide and the C terminal end of the FLAv41E protein were successfully
expressed in Sf-21 insect cells using a baculovirus system. Both constructs were
detected in the cell pellet extracts by Western blot analysis.
The results obtained are discussed with regard to comparative analysis of the three
different approaches used to identify and characterise salivary glands and saliva
molecules for anti-tick vaccine development.