James Bennett - US grants

The adult male Schistosoma mansoni appears to regulate its motor activity by some rather unique mechanisms. One is that it appears to be dependent on environmental Ca++ for normal functioning of its musculature. Another is that the tegument appears to have electrical properties which allow this non-nervous tissue to regulate muscle activity within the parasite. One objective of the proposed research os tp determide to what degree and how the tegument functions in regulating inorganic ion fluxes between the parasite and its environment. This will be accomplished through an analysis, by means of ion selective Ca++ and Na+ electrodes, of the ionic composition of the tegumental cytoplasm, the muscle cytoplasm and interstitial spaces in the animal under control and experimental conditions. These experiments will be complimented with a biochemical study of the parasite's Ca++ pump. A second objective is an examination and characterization of the electrical properties of the tegumental membranes, using electrophysiological techniques, to determine if they are electrically excitable and capable of carrying active propagated potential changes which function to modulate motor activity within the parasite. Results from these studies may point to possibilities for development of agents specifically able to disrupt ion transport or active membrane responses. Such agents may prove to be uniquely effective and specific therapeutic agents against schistosomes.

Our specific aim is to examine the effects of drugs, immune sera (polyclonal and monoclonal) and effector cells upon those processes of immature schistosomes which (1) regulate the parasite's tegumental membrane potential, (2) which are responsible for regulating its motor activity and (3) which regulate the degradation of labeled glucose. Our short term objective is to provide basic data on the major physiological and biochemical events that occur during the development of the schistosome and to use this basic data to help in the analysis of the action of antischistosomal drugs, metabolic inhibitors, lectins, neurotransmitters and surface active agents upon the developing parasite. In addition, we hope to take advantage of the schistosomule's unique responsiveness to immune sera and certain immune effector cells by studying the action of these components on mechanisms which regulate important tegumental and motor functions of the parasite. These studies will be accomplished through electrophysiological, biophysical and biochemical methods that have been developed and published as a result of work in our lab on the adult schistosome. Our long-term objective is to gain a better understanding of the physiology, biochemistry and immunobiology of the developing parasite and to use this information in the design of drugs or vaccines against the larval forms of this parasite.

The disease schistosomiasis is fundamentally due to the immunological reaction of the host to the presence of the parasite's eggs imbedded within its liver. This fact makes viable the concept of eliminating the disease by targeting drugs to destroy the egg producing capability of the female schistosome. This concept has been partially confirmed by previous research but we feel that real progress in this area awaits a clearer understanding of the biochemical mechanisms responsible for egg production. To this end, we have been studying the role which lipids play in regulating egg production with a particular interest in the dolichols, a class of lipids which play a significant role in the glycosylation of proteins. Since the production of these lipids is regulated by the rate-limiting enzyme HMG-CoA reductase, we explored the biochemical and physiological effect of a selective and potent inhibitor of this enzyme called mevinolin. We observed that when mevinolin was given at high doses to infected mice it reduced the appearance of the liver pathology normally induced by the parasite while at lower doses this change was not observed. In fact, worms retrieved from the latter were found to produce 6 times more eggs when placed in culture and compared to worms recovered from nonmevinolintreated mice. When mevinolin is incubated in the presence of cultured parasites it appears to selectively block egg production by the parasite. This drug also inhibits the conversion of 14C-acetate into an isoprenoidlike compound that is necessary for the glycosylation of parasite proteins. Our results are consistent with the idea that the female schistosome's HMGCoA reductase activity is elevated when exposed to mevinolin in vivo and that the activity of this enzyme regulates the production of a nonsterol lipid which in turn regulates the rate at which the female produces eggs. Our immediate goal is to confirm the above hypothesis and then attempt to identify agents that can stop egg production and thus reduce or eliminate the pathology associated with schistosomiasis.

Mevinolin is a specific and potent inhibitor of the rate-limiting enzyme of the mevalonate pathway, HMG-CoA reductase. Mevinolin stops cell growth and can block the action of oncogenes, a group of proteins involved in cell proliferation. Mevinolin stops egg production by Schistosoma mansoni and if the drug is administered chronically into infected mice it will eliminate the infection. The drug's action on schistosomes can be abrogated by mevalonate and farnesol a 15 carbon isoprenoid. The major endproducts of the mevalonate pathway, which consist of cholesterol, dolichols, ubiquinones, hema-a juvenile hormones (in insects) and isopentenyl-tRNA cannot abrogate the effects of mevinolin. The recent discovery that certain oncogenes and other G-binding proteins can be posttranslationally modified on their carboxy terminus with farnesyl (C-15) or geranylgeranyl (C-20) and that the biological activity of these modified proteins depends upon their prenylation has raised the question of their role in schistosomes. Schistosome proteins can be labeled with mevalonate and in particular those having a molecular weight of 25 kDa. These 25 kDa mevalonate labeled proteins bind GTP and the lipid attached to them appears to be geranylgeranyl. At least two prenoid:protein transferase enzymes have been characterized and there are endogenous schistosome proteins which these enzymes can prenylate. Farnesyl protein transferase (FPT) selectively labels 46 kDa schistosome proteins while geranylgeranyl protein transferase (GGPT) labels 25 kDa proteins . Schistosome FPT will farnesylate p2l-Hras while the parasite's GGPT will geranylgeranylate a mammalian protein (rab1b) involved in cell trafficking. From this data we have two major objectives, based upon the working hypothesis that prenylated G-binding proteins are vital for parasite function. Our first objective is to purify one of the 25 kDa prenylated proteins and determine how it functions within the schistosome. Our second objective is to characterize and purify schistosome FPT. Classical protein purification methods will be employed to purify and obtain amino acid sequence data on the 25 kDa prenylated protein. Sequence information from this schistosome protein and from highly conserved regions of sequenced eukaryotic G-bind proteins will be used for the construction of probes for probing cDNA libraries or parasite RNA or genomic DNA using PCR. Cloned genes will be sequenced, expressed and purified. Numerous studies on the recombinant 25 kDa protein will attempt to determine its function. Our second objective will focus on the biochemical characteristics of the schistosome's FPT. The proposed research offers us the opportunity to study the function of a novel group of lipid modified schistosome proteins that probably play a vital role in regulating schistosome reproductive processes as well as other vital functions and serve as potential targets for the development of an antischistosomal drug and/or vaccine.

DESCRIPTION (Adapted from the applicant's abstract): The long-term objective of this project is to develop a rapid, inexpensive and reliable assay to monitor the presence of praziquantel (PZQ) resistant schistosomes in humans. The proposal is a response to recent studies that suggest resistance to PZQ may exist in some parts of Africa, including Egypt, where the PI and colleagues have shown that some villagers infected by schistosomes cannot be cured by 3 doses of the drug. The proposal includes 4 specific aims that are somewhat interdependent: First, they propose to characterize the biological properties of schistosomes that are less susceptible to PZQ both in vivo and in vitro. Second, they will attempt to determine if the apparent resistance to PZQ is genetically transmitted. Third, they will attempt to identify the gene(s) that underlie or are linked to PZQ resistance, and use this information to develop a convenient assay that is capable of detecting resistant isolates using miracidia obtained from eggs shed by infected individuals. Fourth, once this assay is developed, they will examine its utility under field conditions. The proposal states that results obtained from this study, and subsequent epidemiological studies using the assay(s) that evolve from it, will be used to assess the emergence of PZQ resistance, and to assist in the design of future treatment strategies.

Neurobiological processes play a vital role in the survival and reproductive fitness of Schistosoma mansoni, a blood fluke which is the causative agent of schistosomiasis, a medically important human disease in much of the world. Our short term goal is to characterize those neurotransmitter receptors of the parasite that play a direct role in regulating muscle cell activity and in the longrun utilize this information to design antischistosomal drugs. We have developed methods for isolating single muscle fibers from adult schistosomes and have been able, for the first time, to directly analyze the pharmacological nature of receptors on these fibers and then manipulate these fibers, using patch clamp technology, to answer the question of how these receptors regulate ion channel activity on their surface. Furthermore, we are utilizing standard molecular techniques to clone, sequence and express these ion channels. This application is an extension of our previous work and will employ the methods described above. We will focus on two rather unique schistosome receptors, a amidated peptide receptor and a receptor for the amino-acid glutamate. Both of these agents produce dramatic effects upon schistosome muscle fibers but as neurotransmitters in the mammalian host they only function within the central nervous system thus making them attractive targets for drug developers. Our objective is to completely characterize the pharmacological nature of these receptors, determine how they regulate ion conductance across the muscle fiber membrane and the nature of the second messengers which mediate these receptor-induced changes in the permeability of muscle fibers to ions. Once we have completely characterized a receptor, we will clone and sequence it to evaluate its molecular nature and then express the receptor in a surrogate cell to confirm if its pharmacological and physiological properties are identical to those obtained from studies of the receptor in schistosome muscle fibers. Collectively our research on receptors which regulate the excitability of schistosome muscle cells will provide detailed information on macromolecules which play a vital role in regulating parasite behavior.