Robert M. Greenberg - US grants

DESCRIPTION (provided by applicant): Project Summary: Trematode flatworms of the genus Schistosoma are the causative agents of schistosomiasis. One potential physiological target for new anti-schistosoma drugs is the worm's system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, Pgp is the product of the multi-drug-resistance 1 gene, which is amplified and over-expressed in tumor cells that show broad drug resistance. Pgp may also play a role in drug resistance in helminths. A schistosome Pgp cDNA (SMDR2) was sequenced several years ago, but has not been functionally characterized. The long-term goal of this proposal is to dissect the functional role and pharmacological sensitivities of SMDR2 and other schistosome drug transporters. We will adapt a calcein fluorescence assay to examine the substrate and inhibitor specificities of expressed SMDR2 and other transporters. We will also examine the effects of praziquantel, the current antischistosomal of choice, on expression of SMDR2, and whether praziquantel-resistant isolates have altered expression of this or other transporters. Finally, we will examine the effect on the parasite of genetic or pharmacological disruption of SMDR2. The specific aims of the project are to answer the following questions: 1. What are the biochemical properties and substrate specificities of SMDR2 and other schistosome drug transporters expressed in mammalian cells? 2. What is the tissue distribution and developmental profile of SMDR2 and other schistosome multi-drug transporters? 3. Does exposure to agents such as PZQ result in changes in SMDR2 expression or distribution? 4. Do isolates of worms with reduced susceptibility to PZQ show differences in expression levels of SMDR2 or other multidrug transporters? 5. What effects do genetic and pharmacological disruption of SMDR2 have on parasite survival, physiology and pharmacological sensitivities/relevance: Schistosomiasis is a major tropical disease caused by parasitic flatworms called schistosomes. Potential physiological targets for new drugs against schistosomiasis might be the transporters that remove wastes and toxins from schistosome cells. We propose to use several approaches to determine the properties of one such schistosome molecule, P-glycoprotein, which, in vertebrates, is also involved in resistance to a broad array of drugs.

DESCRIPTION (provided by applicant): Trematode flatworms of the genus Schistosoma are the causative agents of schistosomiasis, a tropical parasitic disease with over 200 million people infected worldwide. Praziquantel (PZQ) is the current drug of choice against schistosomiasis, but the mechanism by which PZQ acts remains poorly defined, even several decades following its discovery. Our laboratory has shown that PZQ is likely interacting with a molecular component of schistosome voltage-gated Ca2+ (Cav) channels, specifically a structurally and functionally atypical Cav channel b subunit. This b subunit subtype has been found to date only in platyhelminths;PZQ is not effective against nematodes, and nematode genomes do not contain this variant Cav channel b subunit. This exploratory R21 proposal describes a high-risk/high-potential payoff project to transfer PZQ sensitivity to nematodes by transforming C. elegans with this variant schistosome Cav channel b subunit. We hypothesize that expression of the variant schistosome b subunit in appropriate C. elegans cell types will result in the formation of nematode Cav channels that are sensitive to PZQ. As such, we will place the schistosome b subunit under the control of several different tissue- and cell-specific C. elegans promoters. We will also use conditional promoters to control expression of the gene. If successful, these experiments will offer further compelling evidence that the schistosome variant b subunit is indeed targeted by PZQ, and will also provide the opportunity to acquire important information about the physiological role played by this unique b subunit. Additionally, development of this transgenic model could pave the way for further experiments (as an R01 project) to obtain details of the mode of action of PZQ, including the identification of interacting factors, by exploiting the far more tractable and powerful C. elegans model system. The specific aims of the project are to: 1. Produce transgenic C. elegans lines expressing the schistosome SmCavbvar Cav channel subunit under the control of various promoters. 2. Test transgenic C. elegans lines expressing bvar for sensitivity to PZQ, either in whole worms or in specific tissues. PUBLIC HEALTH RELEVANCE: Schistosomiasis is a major tropical disease caused by parasitic flatworms called schistosomes. Although there is an effective drug against schistosomiasis, the mechanism by which it works is unclear. By understanding that mechanism, it may be possible to obtain drugs that affect the same molecular target, or molecules that interact with that target. In this project, we are using an innovative approach to define the molecular target of this drug with a future goal of identifying other factors that might interact with this drug target.

DESCRIPTION (provided by applicant): Blood flukes, parasitic flatworms of the genus Schistosoma, cause schistosomiasis, a tropical disease that affects hundreds of millions of people worldwide. The current drug of choice against schistosomiasis is praziquantel. Indeed, praziquantel is the only drug currently available in most parts of the world. Such a situation is perilous, particularly in light of reports of emerging parasite resistance to praziquantel. The nee for new therapeutics is therefore urgent. The majority of current anthelmintic drugs target ion channels, validating these proteins as outstanding therapeutic targets. This high-risk, high-payoff project will initiate studies on an entirely unexplored family of schistosome ion channels, the transient receptor potential (TRP) channels. Members of the TRP channel family are strikingly diverse in their activation mechanisms and ion selectivity, but share a common core structure. They are critical to transducing sensory signals, responding to a wide range of external stimuli, and are also involved in regulating levels of intracellular calcium. Mammalian TRP channels are currently under intense investigation as therapeutic targets for treatment of pain, cancer, and a variety of other conditions. We hypothesize that schistosome TRP channels are essential to fulfillment of the schistosome life cycle, which depends on external cues for host-finding and migration to predilection sites within the host, as well as regulation of intracellular calcium. However, the function of these channels in schistosomes and other parasites is entirely unexplored. This project will use parallel strategies to define the roles thee channels play in schistosome physiology, and has the potential to provide novel targets for new antischistosomal agents. The specific aims are to: 1. Determine the effects of genetic and pharmacological disruption of normal TRP channel function on schistosome survival and physiology, and 2. Functionally express and determine the properties and pharmacological sensitivities of a subset of S. mansoni TRP channels. PUBLIC HEALTH RELEVANCE: Schistosomiasis is a major tropical disease affecting hundreds of millions worldwide. It is caused by parasitic flatworms called schistosomes. There is currently only a single drug generally used to treat schistosomiasis, a very treacherous situation. Clearly, new antischistosomal agents are needed. Many of the drugs against parasitic worm infestations target ion channels, which are essential to normal functioning of the parasite's neuromusculature. Thus, ion channels are an excellent choice as potential targets for new anthelmintic drugs. This exploratory project will use a variety of approaches to study a family of schistosome ion channels that have not previously been investigated, with the goal of defining the roles they play in the parasite life cycle and ways to interfere with their function. These studies could eventually lead to new therapeutic strategies against schistosomiasis.

DESCRIPTION (provided by applicant): Schistosomiasis is a devastating, potentially fatal tropical disease that affects hundreds of millions worldwide. In the absence of treatment, schistosome infections are chronic and persistent, often lasting for years or decades, resulting in significant and permanent damage to various organs, severe morbidity, and, in some cases, death. The disease is caused by parasitic flatworms of the genus Schistosoma. Though schistosomes do not replicate within the host, they produce hundreds or thousands of eggs each day. Those eggs that are not excreted remain within the host and evoke an immunopathological response that can lead to chronic disease. How the host immune system responds to schistosome infection determines in large part the balance between protective immunity (health) and immunopathology (morbidity). Several studies have shown that molecules excreted, secreted, or derived from both eggs and worms have potent immunomodulatory properties, but the underlying mechanisms by which these factors are presented to the host have remained elusive. Some of these molecules are potential substrates of ATP binding cassette (ABC) proteins. Members of the ABC superfamily of proteins are efflux transporters associated with the phenomenon of multidrug resistance. In addition to their roles in efflux of drugs and toxins, ABC transporters play critical roles in a wide variety of physiological processes including regulation of immune function. For example, they transport known immunomodulators with high affinity, are involved in antigen presentation, and have been implicated in modulation of immune functions such as T cell migration, T helper cell polarization, and dendritic cell maturation and migration. We hypothesize that schistosomes use a subset of their ABC transporters to mediate excretion, secretion, or presentation of parasite signaling molecules that shape host immune responses and influence pathology. The specific aims of this project will focus on parasite egg-evoked host responses, testing this hypothesis both in vitro (Aim 1) and in vivo (Aim 2). These experiments will provide a unique opportunity to define basic molecular mechanisms underlying polarization of host T-cell responses. Preliminary results we have obtained support our hypothesis, and speak to the feasibility of the proposed experiments. This exploratory project provides a unique opportunity to define basic molecular mechanisms underlying parasite-host interactions, and also holds the promise of offering new therapeutic targets that can be exploited to reduce or eliminate disease pathology.

DESCRIPTION (provided by applicant): Parasitic helminths such as schistosomes and filarial and soil-transmitted nematodes are estimated to infect at least a billion people worldwide, with huge impacts on human health and economic development. In the absence of vaccines, treatment and control of these neglected tropical diseases relies in large part on a small set of anthelmintic drugs. However, diagnosis and monitoring of disease transmission and efficacy of treatment depends almost entirely on methods that are inaccurate, labor-intensive, and unreliable. These limitations are amplified and take on added significance in mass drug administration programs, where measures of effectiveness depend on the ability to accurately monitor treatment success (or failure), changes in disease transmission rates, and emergence of drug resistance. Molecular methods for detection and quantitation of parasite nucleic acids in host fluids or tissues offer reliability, sensitivity, and specificity, but depend on availability f necessary infrastructure, highly-trained staff, and expensive and delicate equipment. The long-term goal of this exploratory project is to overcome these limitations. To do so, we will adapt isothermal molecular assays such as loop-mediated isothermal amplification (LAMP) to a simple, hand-held, point-of-care microfluidic device that allows sensitive and specific detection of helminth parasite nucleic acids in infected hosts. We will use animal models of parasitic helminth infection to demonstrate the feasibility of this technology and as proof of principle. Use of such a device could provide critical advancements in diagnostics, monitoring of disease prevalence and treatment efficacy in mass drug administration programs, and detection of treatment failures that might warrant attention as signs of emerging drug resistance. The specific aims are to: 1) Optimize isothermal amplification assays of helminth nucleic acids in samples from parasite-infected hosts~ and 2) Develop and demonstrate the feasibility of a simple nucleic acid amplification test for parasitic helminths in a point-of-care device format. Translation of this type of simple, inexpensive, self-contained technology for detection of parasitic helminth nucleic acids in hosts or in the environment could ultimately transform analysis of treatment and control options in the developing world.

Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a tropical disease affecting hundreds of millions of people worldwide. There is no vaccine, and only a single drug (praziquantel) available for treatment and control. Many anthelmintics, likely including praziquantel, act on ion channels, membrane protein complexes that are essential for normal functioning of the neuromusculature and other tissues. However, few helminth ion channel families have been assessed for their properties and for their roles in parasite physiology. One such overlooked group of helminth ion channels is the transient receptor potential (TRP) channel superfamily. Members of the TRP channel family are widely diverse in their activation mechanisms and ion selectivity, but share a common core structure. They are critical to transducing sensory signals, responding to a wide range of external stimuli, and are also involved in other functions, such as regulating intracellular calcium and organellar ion homeostasis and trafficking. TRP channels also respond to endogenous agents, including those involved in inflammatory signaling. Our published and preliminary pharmacological and knockdown studies show that schistosome TRP channels can be targeted to impact normal neuromuscular and sensory function. More significantly, they appear to have novel pharmacological sensitivities. Specifically, our results are consistent with the schistosome TRPA channel (SmTRPA) having at least some of the pharmacological sensitivities of mammalian TRPV1 channels, particularly notable as there are no TRPV channels represented in schistosome genomes. Preliminary functional expression studies support this contention. We hypothesize that in schistosomes, SmTRPA fulfills some of the roles of missing TRPV channels. We also hypothesize that SmTRPA and perhaps other schistosome TRP channels regulate critical parasite-host interactions required for successful infection. This project will use parallel strategies to define the roles SmTRPA and other TRP channels play in schistosome biology, including parasite-host interactions, and assess SmTRPA channel function directly. Finally, we hypothesize that the schistosome TRP channel, SmTRPML, plays key roles in schistosome endolysosomal physiology that can impact autophagy and nutrient acquisition. Our studies will elucidate the biological roles and physiological properties of an almost entirely unexplored family of parasite ion channels, information which could in the future be used to provide novel candidate targets for new or repurposed antischistosomal agents. The specific aims of this project are to: 1) Determine the role that SmTRPA and other TRP channels play in the schistosome life cycle, including in parasite-host interactions; 2) Use functional expression to test whether schistosome sensitivity to TRPV1 modulators is mediated specifically by SmTRPA; and 3) Elucidate the role of the schistosome TRPML channel in endolysosomal functions, including nutrient acquisition and autophagy.

Project summary: Infectious diseases that disproportionately impact impoverished regions in the tropics have devastating impacts on human health and economic development, with billions of people at risk. These diseases include HIV/AIDS, malaria, and tuberculosis, but also the neglected tropical diseases (eg, schistosomiasis, filariasis, and other helminth, microbial, and protozoan infections). The agents that cause these diseases are often co-endemic. Concomitant infections can alter host responses, disease prognosis, transmission dynamics, and treatment outcomes, as well as posing difficulties for accurate interpretation of data focused on single infections. A single, simple diagnostic test that could specifically and concurrently detect those infections present in each individual with high sensitivity would be a breakthrough in terms of cost, time, and accurate diagnosis. Unfortunately, current diagnosis and monitoring of disease transmission and efficacy of treatment depend largely on methods that are often inaccurate, labor-intensive, or unreliable. These limitations acquire added significance in mass drug administration programs, where measures of effectiveness require accurate monitoring of infection (and coinfection) status, treatment success, disease transmission rates, and emergence of drug resistance. Molecular detection of pathogen nucleic acids in host fluids or tissues offers reliability, sensitivity, and specificity, but current methods require infrastructural support, highly- trained staff, and expensive, delicate equipment. In this project, we will build upon our substantial advances in development of microfluidic, point-of-care/contact (POC) devices that enable on-site, inexpensive molecular diagnosis of infection and monitoring of disease transmission by minimally-trained personnel. Specifically, we will: Aim 1) develop, optimize, and validate a new 2-stage amplification pathogen detection protocol (RAMP) using benchtop assays; and Aim 2) transfer and adapt the benchtop RAMP assays developed in Aim 1 to a new microfluidic, multiplexing chip, and verify that the chip allows for simultaneous POC detection of multiple pathogen nucleic acids. Accomplishment of these aims will serve as proof of concept and set the stage for more extensive refinement and testing in future work using animal models of infection and coinfection, clinical samples, and field studies. The approach will readily translate into groundbreaking new technology for field-ready, POC molecular diagnosis and monitoring of tropical diseases and coinfections.

Project Summary: Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease affecting hundreds of millions of people worldwide. There is no vaccine, and only a single drug, praziquantel (PZQ), is available for treatment and control. Though indispensable, PZQ has significant shortcomings, and reliance on only one drug for such a highly prevalent disease is dangerous, particularly in light of reports of PZQ-resistant worms. The need for new therapeutics is therefore urgent. Ion channels underlie electrical excitability in cells, and are validated targets for drugs and toxins, including a large number of current anthelmintics. However, only a few parasite ion channel families have been exploited as drug targets. In this project, we focus on one member (SmTRPA) of a largely unexplored family of schistosome ion channels, the transient receptor potential (TRP) channels. TRP channels comprise a diverse family of ion channels that share a common core structure, but are widely varied in their activation mechanisms and ion selectivity. TRP channels are critical to transducing a wide range of sensory signals. They are also involved in a variety of other functions, such as regulation of intracellular and organellar ion homeostasis, and are currently under intense investigation as therapeutic targets for several conditions. We hypothesize that TRP channels from schistosomes and perhaps other parasitic platyhelminths may be excellent targets for new anthelmintics, as they are likely essential for fulfillment of the parasite life cycle, which depends on external cues for host-finding and migration to predilection sites, as well as regulation of neuromuscular activity and ion homeostasis. Our published and preliminary studies show that SmTRPA, the single S. mansoni TRPA1-like channel, can be targeted to impair normal neuromuscular function. Notably, SmTRPA also exhibits at least some of the pharmacology of another type of TRP channel, TRPV1. This mixed TRPV1/TRPA1 pharmacology is particularly intriguing, as TRPV channels are not represented in schistosome (or other parasitic platyhelminth) genomes. Preliminary functional expression studies using Ca2+ imaging are consistent with this interpretation. In this exploratory project, we will use calcium imaging in a high-throughput screen to identify small molecules that selectively activate this pharmacologically atypical TRP channel (Aim 1). This aim will provide lead scaffolds for further characterization and refinement. In Aim 2, we will test responses of schistosomes at different life-cycle stages to known and newly-identified SmTRPA modulators, measuring parameters such as worm survival, motility, male-female pairing, and egg production. This aim will provide a deeper understanding of the effects of known SmTRPA modulators, and assess newly-identified SmTRPA modulators for antischistosomal activity. This project could eventually lead to development of new antischistosomals that target a novel receptor, and could serve as a model for discovery of other anthelmintics that target TRP channels from other parasitic pathogens.

Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease affecting hundreds of millions of people worldwide. There is no vaccine, and only a single drug (praziquantel; PZQ) available for treatment and control. Although PZQ was introduced decades ago, its mode of action is still not completely understood. However, one major effect of PZQ is to disrupt the tegument of the worm, likely resulting in exposure of parasite antigens that are normally hidden from the host. The effectiveness of PZQ drops significantly in immunocompromised mice, and it has been suggested that host antibodies act directly against parasite antigens that become exposed on the worm surface as a result of this PZQ-induced tegumental disruption. We have been using RNAseq to investigate transcriptomic changes that occur in schistosomes following administration of PZQ to S. mansoni-infected mice. We find that in response to low-dose PZQ (100 mg/kg), adult worms of each of 3 different S. mansoni strains dramatically increase expression (up to ~150-fold) of MEG-3 transcripts. MEG-3 is one of a group of micro-exon genes (MEG) found within the S. mansoni genome. MEGs have the potential to generate high levels of protein variation through alternative splicing of short, symmetric exons organized in tandem. Most of these proteins appear to be secreted by the worm, and others have postulated that expression of such a complex pool of variant MEG proteins could constitute a schistosome immune evasion mechanism. We hypothesize that in response to PZQ-dependent immune attack, schistosomes activate immune evasion/decoy mechanisms, one of which is increased expression of MEG-3 proteins (and associated transcripts). This exploratory project will test predictions of this hypothesis. We will compare expression of MEG-3 transcripts following low-dose PZQ in worms in contact with an intact immune system versus those in contact with a defective immune system (eg, infecting immunocompromised mice). We will also test whether other antischistosomal drugs that do not require an intact host immune system for activity do not induce upregulation of MEGs, and we will determine whether reconstitution of the immune system of an infected immunocompromised host with serum from schistosome-infected immune-intact mice can restore the PZQ-induced upregulation of MEG-3. These studies will lay the groundwork for a future R01 proposal that will examine the role of adaptive schistosome defenses in parasite drug susceptibility and long-term survival. This work could ultimately lead to novel therapeutic strategies for targeting parasite immune evasion mechanisms as a means to increase drug susceptibility or disrupt the schistosome life cycle.

Project summary: Schistosomiasis, a neglected tropical disease affects hundreds of millions of people worldwide and is associated with significant morbidity and mortality and imposes a high socioeconomic burden on many affected developing countries. Schistosomiasis is caused by water-borne parasitic trematodes of the genus Schistosoma, which can survive inside human hosts for decades. Disease pathology results from deposition of schistosome eggs in host tissues, which evokes an immunopathological response from the host. Recent work shows that oncolytic viruses with limited host range in mammals can eliminate cancer cells without harming the human host and these viruses are being actively investigated for potential clinical use. We recently performed preliminary studies showing that one of these oncolytic viruses is capable of infecting schistosomes and replicating within the parasites. Thus, exposure of Schistosoma mansoni to the virus in vitro results in 100% infection of worms, resulting in tegumental disruption, damage to other worm tissues, and parasite lethality. Unlike praziquantel, the current drug used to treat and control schistosomiasis, this oncolytic virus impacts all intramammalian parasite stage including schistosomula, juvenile, and adult schistosomes. This innovative exploratory proposal will confirm and extend these preliminary studies with the goals of determining the dynamics of infection and whether this oncolytic virus can be used to infect and potentially kill schistosomes in vivo without harming the host. Specifically, we will Aim 1: Determine the effects of oncolytic virus on the different life stages of schistosomes in vitro and Aim 2: Determine the effects of oncolytic virus on schistosomes within the mammalian host. We have excellent interdisciplinary collaborators to carry this project successfully. Accomplishment of these aims will serve as proof of concept and set the stage for more extensive refinement and testing in future work using animal models of infection and co-infection to understand the underlying parasite pathways and target the pathway hijacked by the virus, perhaps leading to new therapeutic targets. These studies will provide a platform to potentially use this oncolytic virus as a ?schisto-lytic? agent due to its extremely narrow host range and could provide an effective safe schistosomiasis therapeutic. The approach will readily translate into groundbreaking new technology for treatment and control of schistosomiasis.