Modulated Enzyme Expression in Gastrointestinal Nematodes

Gastrointestinal nematodes cost the farming industry millions of pounds a year. Control is largely dependent on the use of anthelmintics but widespread resistance to these drugs is now developing. Attention has, therefore, turned to the development of novel methods for parasite control. Recently, research has focused on the identification of parasite antigens which stimulate protective host immune responses with the aim of vaccine development. Parasite enzymes are appropriate targets for this strategy but there is some evidence that parasites can 'adapt' enzyme expression in response to host immune attack while the degree to which enzyme expression is influenced by parasite strain divergence is largely undefined. These factors could have considerable implications for the development of subunit vaccines and novel anthelmintics. The first part of this study characterised the proteinases of the parasitic stages of Teladorsagia circunicincia, an important ovine abomasal nematode in the UK. Proteinases released during the in 14tro maintenance of third or fourth stage larval as well as adult parasites were characterised on the basis of pH optima, molecular size and specific proteinase inhibitor sensitivity. Proteinases were released in a stage specific manner and metallo-proteinases predominated with the ability to degrade a variety of potential natural protein substrates. Immunological studies indicated that these proteinases were weakly immunogenic stimulating enzyme inhibiting antibody responses. Enzyme expression in the ovine abomasal parasite Haeinonchus conforms, including proteinases and superoxide dismutases, was examined in comparisons of 1) UK and Australian strains of the parasite and 2) parasites surviving in lambs vaccinated against homologous challenge, the vaccine being an integral gut membrane protein complex, and those from the corresponding challenge controls. In both comparisons marked differences in the level of total proteinase expression were noted. The influence of the host on parasite enzyme expression was studied more closely in the rat/Nippostrongylus brasiliensis intestinal nematode infection system. Profound differences were demonstrated in the proteinase content of adult worm extracts, as judged by gelatin-substrate gel analysis, when parasites harvested from individual rats were compared following primary infection. Moreover, the intra-host environment also markedly influenced parasite superoxide dismutase isoenzyme expression. Similar effects were noted when worm populations harvested following primary and secondary infections were compared. To facilitate the definition of the molecular events underlying variable parasite enzyme expression, a reverse transcriptase polymerase chain reaction (RT-PCR) assay for quantifying total SOD mRNA transcript production in N. brasiliensis was developed. N. brasiliensis SOD encoding cDNAs were isolated and their nucleotide sequences determined, these analyses indicating that sequence variants were contiguously expressed in the adult parasite. The RT-PCR assay was developed using primers designed to anneal to all the known SOD transcripts and then used to quantify SOD encoding tnRNA production in a variety of different parasite populations and these data compared with total enzyme activity determinations. Highly significant correlations were obtained indicating that SOD mRNA transcript levels were indicative of active enzyme production. In conclusion, the work described in this thesis provided further evidence to support the view that parasites can modulate enzyme expression in response to the changing intra-host environment. The development of an RT-PCR approach with the potential to quantify enzyme expression in a gene specific manner will facilitate experiments to define whether or not altered enzyme expression is primarily due to genotypic or phenotypic variation in the parasite population and provides the sensitivity to examine individual worms