Penn Veterinary Medicine | University of Pennsylvania

Description of Research Expertise

My laboratory is attempting to better understand the molecular components of the neuromuscular system of human blood flukes such as Schistosoma mansoni. Schistosomes are parasitic flatworms that cause schistosomiasis. Approximately one sixth of the world's population is at risk for this disease, and hundreds of millions of people worldwide are infected with these parasites. A major focus of my research is on the structure and function of schistosome voltage-gated Ca2+ (Cav) channels. Our evidence indicates that praziquantel (PZQ), the current drug of choice against schistosomiasis, may be interacting with schistosome Cav channel subunits. Schistosome Cav channels may also provide targets against which new, very potent and specific antiparasitic agents may be targeted. More recently, we have been investigating the role of schistosome multidrug resistance proteins in parasite physiology, and the interaction of these transporters with PZQ.

Cav channel work:
Cav channels are heteromultimeric proteins that open in response to depolarization of the cell membrane, allowing Ca2+ ions to flow down the electrochemical gradient and into the cell. They consist of the pore-forming alpha-1 subunit and modulatory subunits such as ß and alpha2-delta. Cav channels contribute to impulse propagation, and, because they regulate intracellular levels of Ca2+, they are also critical for coupling cellular excitation to Ca2+-dependent cellular responses. Different Cav channel subtypes have specific expression patterns and physiological and pharmacological properties.
A major project in my laboratory is to study the structure and function of Cav channels in schistosomes. The differences in primary amino acid sequence that have arisen throughout evolution between the channels of these early invertebrates and those of mammals may have important functional consequences. These differences may aid in understanding the evolution of voltage-gated ion channels and the structure/function relationships within and between channel proteins. Furthermore, information regarding the schistosome channels may eventually lead to their use as targets for new rationally designed and very specific anthelmintic agents.
Of particular interest in this regard is the action of PZQ, the current drug of choice against schistosomiasis. Although the precise mode of action of PZQ is unknown, one of the initial responses to the drug is a disruption of Ca2+ homeostasis in the worm, perhaps involving interaction with Cav channels. Our results indicate that components of schistosome Cav channels are in fact sensitive to PZQ. Indeed, an atypical Cav channel ß subunit that is found only in S. mansoni, S. japonicum, and other platyhelminths can confer PZQ sensitivity to an otherwise insensitive mammalian Cav channel alpha-1 subunit. These schistosome ß subunits have very unusual structural features, including the absence of two conserved consensus protein kinase C (PKC) phosphorylation sites in the beta interaction domain (BID), a highly conserved, structurally important region of the ß subunit. Creation by mutagenesis of either or both of these PKC sites in the BID of these ß subunits eliminates the ability of this subunit to confer PZQ sensitivity. Elimination of these PKC sites in a mammalian b subunit results in a b subunit that can now confer PZQ sensitivity to a mammalian Cav channel. Thus, absence of PKC phosphorylation in the BID of ß subunits appears to be a critical factor in the action of PZQ. Interestingly, platyhelminths also express a conventional ß subunit, which does not confer PZQ sensitivity to Cav channels, but which has other unique properties that we are currently exploring.
We plan to continue investigating the role of BID phosphorylation, using biochemical, molecular, and electrophysiological approaches. We also plan to use localization and expression of schistosome alpha-1 and ß subunits to determine which subtype combinations are found in vivo, and which are involved in PZQ sensitivity in the parasite. We also plan to explore the consequences of genetic knockdown or aberrant expression of the unusual b subunit on schistosomes and on PZQ susceptibility. Our ultimate goals are to obtain a clearer understanding of the mode of action of PZQ, the mechanisms by which resistance to PZQ may arise, and the physiological role of Cav channels in schistosomes and other platyhelminths.

Multidrug resistance in schistosomes:
We have also been exploring the properties and substrate sensitivities of a schistosome homolog of P-glycoprotein (Pgp), a protein associated with multidrug resistance. These proteins are transporters which pump metabolites, toxins, and xenobiotics out of the cell. In addition to their role in drug resistance, they also function in normal physiological excretion of toxic wastes from cells.
We have cloned and expressed a schistosome Pgp homolog in CHO cells, and used a fluorescence assay (calcein AM) to monitor the function and pharmacological sensitivities of this transporter. The assay appears to work well, and we are currently working to obtain stable cell lines in which all cells express schistosome Pgp, a requirement for more precise interpretation of data. In addition, we have found that exposure of adult male parasites to a sublethal concentration of PZQ results in an upregulation of schistosome Pgp expression at both the RNA and protein levels. Thus, Pgp and other members of this class of proteins may be involved in metabolism of PZQ by schistosomes. Alternatively, since PZQ has been shown by others to be a potent inhibitor of mammalian Pgp, it is possible that Pgp is upregulated to counteract the inhibitory effects of PZQ on normal schistosome excretory physiology. In either case, these results indicate that Pgp and other multidrug resistance proteins may prove to be useful targets for new antischistosomals, or for drugs that potentiate the action of PZQ. We also have preliminary evidence indicating that schistosome Pgp is upregulated in a schistosome isolate from Egypt that shows reduced susceptibility to PZQ. Thus, in addition to its likely interaction with PZQ during acute exposure to the drug, Pgp may also be involved in the development of drug resistance in schistosomes.
We plan to continue this work, exploring the role of Pgp and other multidrug resistance proteins in schistosome physiology and drug susceptibility, using the calcein AM and other assays of protein function, as well as genetic disruption by RNAi. An NIH R01 grant supporting this project was funded in April, 2007.

Other projects:
We have also been exploring the properties of other schistosome receptors, channels, transporters, and signaling systems. In particular, we have focused on the nitric oxide (NO) signaling system in adult worms. NO is a gas involved in intercellular signaling, defense, and other functions. In schistosomes, it is also likely to be playing an important role in parasite-host interactions, as NO cytotoxicity may be used by the worm for non-immune defense against host responses to infection. Using immunological, histochemical, and other methods, we provided evidence for the presence and distribution of two isoforms of schistosome nitric oxide synthase, the enzyme that synthesizes NO. We have also described the distribution and pharmacological sensitivity of authentic NO in living schistosomes, using a fluorescent NO indicator, and we are currently exploring downstream responses to NO, including changes in gene expression. Ultimately, we intend to define agents that interfere with these schistosome NO pathways and, as a consequence, might disrupt the worm's life cycle as well as its defense mechanisms against host responses.
Finally, we have also been investigating other schistosome receptors and channels (eg, TRP channels, peptide-gated channels), which have potential to serve as targets for novel anthelmintics.

Selected Publications

Messerli SM, Kasinathan RS, Morgan W, Spranger S, Greenberg RM: Schistosoma mansoni P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility. Mol Biochem Parasitol. 167: 54-59, 2009.

Kasinathan, R., Goronga, T., Messerli, S.M., Webb, T.R., and Greenberg, R.M.: Modulation of a Schistosoma mansoni multidrug transporter by the antischistosomal drug praziquantel. FASEB Journal, in press 2009.

Salvador-Recatala, V., Schneider, T., and Greenberg, R.M. : Atypical properties of conventional and variant Ca2+ channel Beta subunits from Schistosoma mansoni. BMC Physiology 8: 6, 2008.

Greenberg, R.M. : Molecular target of the antischistosomal drug praziquantel. Future Microbiology 2: 265-268, 2007.

Messerli, S.M., Birkeland, S.R., Bernier, J., Cipriano, M.J., Morgan, W., McArthur, A.G., and Greenberg, R.M. : Nitric oxide-dependent changes in Schistosoma mansoni gene expression. Mol. Biochem. Parasitol 150: 367-370, 2006.

Jeziorski, M.C. and Greenberg, R.M.: Voltage-gated Ca2+ channel subunits from schistosomes and other platyhelminths: potential role in praziquantel action. Int. J. Parasitol. 36: 625-632, 2006.

Kohn, A.B., Lea, J., Moroz, L.L, and Greenberg, R.M. : Schistosoma mansoni: Use of a fluorescent indicator to detect nitric oxide and related species in living parasites. Experimental Parasitology 113: 130-133, 2006.

Greenberg, R.M.: Praziquantel: Mechanism of action. In: "Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology and Physiology" CABI,Oxfordshire, UK Page: 269-281, A. Maule, Marks, N.J. (eds.). 2006.

Greenberg, R.M.: Ca2+ signaling, voltage-gated Ca2+ channels, and praziquantel in flatworm neuromusculature. Parasitology 131: S97-S108, 2005.

Greenberg, R.M. : Are Ca2+ channels targets of praziquantel action? Int. J. Parasitol. 35: 1-9 , 2005.