Abstract:
Tick proteins functioning in vital physiological processes such as blood meal uptake,
digestion and reproduction are potential targets for anti-tick vaccines, since vaccination
could disrupt these essential functions and ultimately affect tick survival. In this study we
identified metzincin metalloproteases from R. microplus, the world’s most economically
important external ectoparasite of cattle, as potential vaccine candidates since they are
implicated to be essential to blood-cavity formation, bloodmeal digestion and
reproduction in ixodid ticks. A vaccine derived from a single member of such a large
family pose the obstacle of redundancy within the family, that may allow the function of
the targeted family member to be taken up by other family members. Therefore the aim
of this study was not only to focus on the physiological importance of each metzincin
transcript, but also to investigate the differential gene expression network between the
different metzincin family members.
Eight transcripts encoding proteins containing the characteristic metzincin zinc-binding
motif HEXXHXXG/NXXH/D and a unique methionine-turn were identified from native
and in-house assembled R. microplus Expressed Sequence Tag databases. These
were representative of five reprolysin-like and three astacin-like metzincin
metalloproteases. Reverse transcription-PCR indicated that the reprolysins were most
abundantly expressed in the salivary glands, whereas the astacins were most abundant
in the midgut and ovaries. In vivo gene silencing utilizing RNA interference, was
performed to assess a possible phenotype in silenced adult female R. microplus ticks
during blood feeding and reproduction. RNAi against two reprolysins and one astacin
significantly affected average egg weight as well as the oviposition rate. Moreover,
integrated real time-PCR studies revealed an extensive cross organ network between
the R. microplus metzincin transcripts, supporting the use of a combinatorial metzincinbased
anti- R. microplus vaccine targeting multiple members of the large metzincin clan
simultaneous. To conclusively evaluate the vaccination potential of the three identified
metzincin candidates, the immunogenicity and protective properties of the recombinant
proteins needs to be determined. Due to metalloproteases destructive activity and
characteristics such as cysteine rich domains, only selected domains of the three
candidates were expressed, using a cost effective Eshericia coli based expression
system. Finally, the ability of each successfully expressed domain to elicit an immune
response and serve as a protective antigen against R. microplus will be screened during
vaccination trials in cattle.
