Remodeling of protein and mRNA expression in Leishmania mexicana induced by deletion of glucose transporter genes.

Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of Leishmania mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose.

An expression system to screen for inhibitors of parasite glucose transporters.

Chemotherapy of parasitic protists is limited by general toxicity, high expense and emergence of resistance to currently available drugs. Thus methods to identify new leads for further drug development are increasingly important. Previously, glucose transporters have been validated as new drug targets for protozoan parasites including Plasmodium falciparum, Leishmania mexicana and Trypanosoma brucei. A recently derived glucose transporter null mutant (Deltalmgt) of L. mexicana was used to functionally express various heterologous glucose transporters including those from T. brucei THT1, P. falciparum PfHT and human GLUT1-resulting in recovery of growth of the Deltalmgt null mutant in glucose replete medium. This heterologous expression system can be employed to screen for compounds that retard growth by inhibiting the expressed glucose transporter. The ability of this expression system to identify specific glucose transporter inhibitors was demonstrated using 3-O-undec-10-enyl-d-glucose, a previously described specific inhibitor of PfHT.

Cells in the vomeronasal organ express odorant receptors but project to the accessory olfactory bulb.

Recent evidence indicates that the vomeronasal organ (VNO) of mice not only responds to pheromones but also to odorants. To analyze whether genes encoding odorant receptors (ORs) are expressed in the VNO, reverse transcriptase-polymerase chain reaction analyses were performed. These led to the identification of 44 different OR genes, comprising class-I and class-II receptors. The genes encoding these receptors were scattered over several gene clusters. The respective OR genes were concomitantly expressed in cells of the main olfactory epithelium (MOE). Although the cells in the MOE were zonally distributed, no such patterns were displayed in the VNO. Cells expressing ORs in the VNO were positive for the TRP2-channel and Galphai, a marker for vomeronasal neurons of the apical layer. In transgenic mice, which coexpress histological markers with the receptor mOR18-2, characteristic morphological differences between cells expressing this receptor in the VNO compared with the MOE became evident. Visualizing the axonal processes of VNO cells expressing distinct ORs revealed that they project to the accessory olfactory bulb (AOB). Axon fibers were visible exclusively in the anterior subdomain; here, they converged into glomerular-like structures positioned at the very rostral tip of the AOB. The findings that a set of ORs is expressed in cells located in the apical layer of the VNO with typical features of VNO sensory neurons that project their axons to the anterior part of the AOB suggest that this population of sensory cells may be considered as a unique facet of the complex chemosensory system.