Similarities and differences in the biotransformation and transcriptomic responses of Caenorhabditis elegans and Haemonchus contortus to five different benzimidazole drugs.

We have undertaken a detailed analysis of the biotransformation of five of the most therapeutically important benzimidazole anthelmintics - albendazole (ABZ), mebendazole (MBZ), thiabendazole (TBZ), oxfendazole (OxBZ) and fenbendazole (FBZ) - in Caenorhabditis elegans and the ruminant parasite Haemonchus contortus. Drug metabolites were detected by LC-MS/MS analysis in supernatants of C. elegans cultures with a hexose conjugate, most likely glucose, dominating for all five drugs. This work adds to a growing body of evidence that glucose conjugation is a major pathway of xenobiotic metabolism in nematodes and may be a target for enhancement of anthelmintic potency. Consistent with this, we found that biotransformation of albendazole by C. elegans reduced drug potency. Glucose metabolite production by C. elegans was reduced in the presence of the pharmacological inhibitor chrysin suggesting that UDP-glucuronosyl/glucosyl transferase (UGT) enzymes may catalyze benzimidazole glucosidation. Similar glucoside metabolites were detected following ex vivo culture of adult Haemonchus contortus. As a step towards identifying nematode enzymes potentially responsible for benzimidazole biotransformation, we characterised the transcriptomic response to each of the benzimidazole drugs using the C. elegans resistant strain CB3474 ben-1(e1880)III. In the case of albendazole, mebendazole, thiabendazole, and oxfendazole the shared transcriptomic response was dominated by the up-regulation of classical xenobiotic response genes including a shared group of UGT enzymes (ugt-14/25/33/34/37/41/8/9). In the case of fenbendazole, a much greater number of genes were up-regulated, as well as developmental and brood size effects suggesting the presence of secondary drug targets in addition to BEN-1. The transcriptional xenobiotic response of a multiply resistant H. contortus strain UGA/2004 was essentially undetectable in the adult stage but present in the L3 infective stage, albeit more muted than C. elegans. This suggests that xenobiotic responses may be less efficient in stages of parasitic nematodes that reside in the host compared with the free-living stages.

The response of monocyte derived dendritic cells following exposure to a nematode larval carbohydrate antigen.

The glycolipid CarLA (carbohydrate larval antigen) is present on the epicuticle of the infective-stage larvae of gastrointestinal nematode parasites infecting livestock. The molecule is lost from the surface of the larvae in the few days post-ingestion by a host animal, and the resulting anti-CarLA antibody response has been demonstrated to be protective in vivo. Both the anti-CarLA response, and anti-parasite immunity in general, are slow to develop, and several months of natural exposure to ingested larvae is required. The current study was designed to provide information on how the anti-CarLA response develops, and focuses on the initial recognition of the molecule by human monocyte derived dendritic cells (mdDC) in vitro. Immunofluorescence and flow cytometry demonstrated that mdDC recognise and internalise both the purified and the native form of CarLA, in the case of the latter once it is shed from the larval surface. However, the recognition of CarLA did not result in classical maturation of DC, while there was only transient or minor up-regulation of CD86, CD83, HLA-DR and CD40. Exposure of mdDC to purified CarLA resulted in the increased production of the pro-inflammatory cytokines IL-6 and to a lesser extent of IL-8 and TNF-α, and a reduced production of the anti-inflammatory cytokine IL-1RA. CarLA therefore has little ability to mature and functionally alter monocyte derived dendritic cell function.

Differential expression of the whey acidic protein gene during lactation in the brushtail possum (Trichosurus vulpecula).

The whey acidic protein (WAP) is a whey protein found in the milk of a number of species. We have isolated and characterised a WAP cDNA clone from the brushtail possum (Trichosurus vulpecula) and examined its expression in the mammary gland. The amino acid sequences of WAP from the possum and another marsupial, the tammar wallaby, share 69% identity, however, less sequence identity exists between the marsupial and eutherian WAP sequences (30-37%). The possum and tammar WAP genes consist of three four-disulphide core (4-DSC) domains, with a WAP motif at the beginning of each domain. In contrast, the eutherian WAP sequences consist of two 4-DSC domains with the WAP motif only present in the second domain. This WAP motif is also present in a number of protease inhibitors found in a wide range of species. Phylogenetic analysis of marsupial and eutherian WAP sequences suggests that the ancestral WAP gene has three domains and that one of the domains has been deleted from the eutherian gene. The profile of WAP gene expression in the possum mammary gland changed throughout lactation, with WAP mRNA levels reaching a peak between days 106 and 177 of lactation. The level of WAP mRNA in the mammary gland appeared to be correlated with the level of circulating prolactin in the lactating female and was different to that observed for several other whey protein genes. Overlapping expression of the WAP and early lactation protein genes, both of which are putative protease inhibitors, may provide protection of milk immunoglobulins that are required for the prolonged period of passive immune transfer to the marsupial pouch young.

Cloning and expression of the transferrin and ferritin genes in a marsupial, the brushtail possum (Trichosurus vulpecula).

Transferrin and ferritin cDNAs have been isolated and characterised from the common brushtail possum (Trichosurus vulpecula), the first marsupial examples of these genes. The transferrin cDNA encodes a 711 amino acid pre-protein which shows high levels of amino acid identity with eutherian transferrins (58-60%) and lactoferrins (54-56%). Phylogenetic analysis suggests that the possum transferrin has evolved independently along a pathway distinct from that of the eutherian transferrins and lactoferrins. Possum H-ferritin is a 182 residue protein which shares 86-94% amino acid identity with mammalian, avian and amphibian sequences. Ferritin mRNA was detected in all tissues tested, whereas transferrin was highly expressed in possum liver and mammary gland, and at lower levels in heart, testis and lung. In the possum mammary gland, ferritin mRNA was expressed throughout lactation with higher levels during the first 30 days which coincides with the high iron concentration of milk at this time. The transferrin gene was differentially expressed during lactation with peak mRNA levels detected during the first 6 days of lactation and after day 106 throughout late lactation. The pattern of transferrin mRNA expression in the mammary gland was identical to that of another whey protein, the late lactation protein, suggesting that the transcription of these genes may be regulated by a similar mechanism in this tissue.