Lisa Sharling - Academia.edu (original) (raw)

Papers by Lisa Sharling

The aim of this research was to identify the erythrocyte surface antigens of P. falciparum-irffec... more The aim of this research was to identify the erythrocyte surface antigens of P. falciparum-irffected erythrocytes (lEs) that are expressed following selection of the parasite for adhesion to the placental receptor chondroitin sulphate A (CSA). These surface antigens are considered potential vaccine targets against pregnancyassociated malaria (PAM). Typically P. falciparum surface antigens are highly sensitive to treatment of the IE with the protease trypsin; however, variant surface antigen (VSA) mediated adhesion to CSA has often been found to be relatively trypsin-resistant. In this study the protease sensitivity of the VSA of CSA binding lEs was determined with regard to serum IgG recognition and placental receptor binding. Discordance between the protease sensitivity for these phenomena was identified; this may have implications for PAM vaccine design. The completion of several Plasmodium genome sequences and advances in mass spectrometry have opened up the field of proteomics t...

The American journal of tropical medicine and hygiene, Jan 5, 2018

In 2004, there existed limited tuberculosis (TB) research capacity in the country of Georgia. In ... more In 2004, there existed limited tuberculosis (TB) research capacity in the country of Georgia. In response, a collaborative research training program (RTP) supported by a National Institutes of Health Fogarty International Center Global Infectious Diseases grant was formed between a U.S. academic institution and the National Center for Tuberculosis and Lung Disease (NCTLD) and other institutions in Georgia. We sought to assess outcomes of this RTP. The TB RTP combined didactic and mentored research training for Georgian trainees. Long-term trainees were supported for a 2-year period and with posttrainee career development mentoring. Metrics used to measure program performance included publications, grants received, and career advancement. From 2004 to 2015, 20 trainees participated in the program with 15 (75%) authoring a total of 65 publications in PubMed-listed journals. The median number of publications per trainee was six (interquartile range 2-14). A total of 16 (80%) trainees r...

Malaria journal, Jan 6, 2004

The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endo... more The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM). Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for a...

Molecular Approaches toward Targeted Drug Development, 2011

... The vicious cycle of diarrhea and malnutrition imperils the health of infants and small child... more ... The vicious cycle of diarrhea and malnutrition imperils the health of infants and small children in the impoverished regions of the ... in mouse microsomes – a problem which must clearly be overcome if these compounds are to advance into a mouse model of cryptosporidiosis. ...

The Journal of Cell Biology, 2001

igh molecular weight homologues of gp91 phox, the superoxide-generating subunit of phagocyte nico... more igh molecular weight homologues of gp91 phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91 pho x domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. el-H egans . RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di-and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed crosslinking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix. *Abbreviations used in this paper: Ce-Duox, Caenorhabditis elegans Duox; dsRNA, double-stranded RNA; FAD, flavin adenine dinucleotide; h-Duox, human Duox; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate; RACE, rapid amplification of cDNA ends; RNAi, RNA interference; TMB, 3,3 Ј ,5,5 Ј -tetramethylbenzidine.

PLoS Neglected Tropical Diseases, 2010

Background: The protozoan parasite Cryptosporidium parvum is responsible for significant disease ... more Background: The protozoan parasite Cryptosporidium parvum is responsible for significant disease burden among children in developing countries. In addition Cryptosporidiosis can result in chronic and life-threatening enteritis in AIDS patients, and the currently available drugs lack efficacy in treating these severe conditions. The discovery and development of novel anti-cryptosporidial therapeutics has been hampered by the poor experimental tractability of this pathogen. While the genome sequencing effort has identified several intriguing new targets including a unique inosine monophosphate dehydrogenase (IMPDH), pursuing these targets and testing inhibitors has been frustratingly difficult.

Molecular and Cellular Biology, 2000

A large number of neuron-specific genes characterized to date are under the control of negative t... more A large number of neuron-specific genes characterized to date are under the control of negative transcriptional regulation. Many promoter regions of neuron-specific genes possess the repressor element repressor element 1/neuron-restrictive silencing element (RE1/NRSE). Its cognate binding protein, REST/NRSF, is an essential transcription factor; its null mutations result in embryonic lethality, and its dominant negative mutants produce aberrant expression of neuron-specific genes. REST/NRSF acts as a regulator of neuronspecific gene expression in both nonneuronal tissue and developing neurons. Here, we shown that heterologous expression of REST/NRSF in Saccharomyces cerevisiae is able to repress transcription from yeast promoters engineered to contain RE1/NRSEs. Moreover, we have taken advantage of this observation to show that this repression requires both yeast Sin3p and Rpd3p and that REST/NRSF physically interacts with the product of the yeast SIN3 gene in vivo. Furthermore, we show that REST/NRSF binds mammalian SIN3A and HDAC-2 and requires histone deacetylase activity to repress neuronal gene transcription in both nonneuronal and neuronal cell lines. We show that REST/NRSF binding to RE1/NRSE is accompanied by a decrease in the acetylation of histones around RE1/NRSE and that this decrease requires the N-terminal Sin3p binding domain of REST/NRSF. Taken together, these data suggest that REST/NRSF represses neuronal gene transcription by recruiting the SIN3/HDAC complex.

Molecular and Biochemical Parasitology, 2004

Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo... more Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo-capping with in vitro recombination and directional cloning of cDNA inserts into a plasmid vector that expresses sequences as thioredoxin fusion proteins. A novel procedure has also been developed for the rapid identification of seropositive clones on high-density filters, using direct labelling of P. falciparum immune immunoglobulin with fluorescein isothiocynate (FITC). This approach combines the advantages of recombination-assisted cDNA cloning with high throughput, non-radioactive serological screening of expression libraries. Production of replicate colony matrices allows the identification of antigens recognised by different pools with different specificities from residents of a malaria endemic region. Analyses of DNA sequences derived from sero-reactive colonies indicate that this is an effective method for producing recombinant proteins that react with antibodies from malaria-exposed individuals. This approach permits the systematic construction of a database of antigenic proteins recognised by sera from malaria-exposed individuals.

Malaria Journal, 2007

Background: Sensitive detection of parasite surface antigens expressed on erythrocyte membranes i... more Background: Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites.

Life Sciences, 1999

The specific cellular response to muscarinic receptor activation is dependent upon appropriate ex... more The specific cellular response to muscarinic receptor activation is dependent upon appropriate expression of each of the five muscarinic receptor genes by individual cells.

Journal of Medicinal Chemistry, 2012

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine.... more Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5'-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high-throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC(50) < 2 nM) against CpIMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model.

Journal of Biological Chemistry, 2010

Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised... more Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k cat ‫؍‬ 0.28 s ؊1 and K m,ATP ‫؍‬ 140 M are similar to those of human TK1, the value of K m (thymidine) ‫؍‬ 48 M is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.

Chemistry & Biology, 2008

Cryptosporidium parvum is an important human pathogen and potential bio-terrorism agent. No vacci... more Cryptosporidium parvum is an important human pathogen and potential bio-terrorism agent. No vaccines exist against C. parvum, the drugs currently approved to treat cryptosporidiosis are ineffective, and drug discovery is challenging because the parasite cannot be maintained continuously in cell culture. Mining the sequence of the C. parvum genome has revealed that the only route to guanine nucleotides is via inosine-5′-monophosphate dehydrogenase (IMPDH). Moreover, phylogenetic analysis suggests that the IMPDH gene was obtained from bacteria by lateral gene transfer. Here we exploit the unexpected evolutionary divergence of parasite and host enzymes by designing a high throughput screen to target the most diverged portion of the IMPDH active site. We have identified four parasite-selective IMPDH inhibitors that display antiparasitic activity with greater potency than paromomycin, the current "gold standard" for anticryptosporidial activity.

Cellular Microbiology, 2007

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family... more The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a > 100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum bloodstage parasites and are exposed on the surface of haemolymph-and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

Bioorganic & Medicinal Chemistry Letters, 2012

Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. p... more Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. parvum and C. hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazolebased inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro.

International Journal of Women's Health, 2015

The aim of this research was to identify the erythrocyte surface antigens of P. falciparum-irffec... more The aim of this research was to identify the erythrocyte surface antigens of P. falciparum-irffected erythrocytes (lEs) that are expressed following selection of the parasite for adhesion to the placental receptor chondroitin sulphate A (CSA). These surface antigens are considered potential vaccine targets against pregnancyassociated malaria (PAM). Typically P. falciparum surface antigens are highly sensitive to treatment of the IE with the protease trypsin; however, variant surface antigen (VSA) mediated adhesion to CSA has often been found to be relatively trypsin-resistant. In this study the protease sensitivity of the VSA of CSA binding lEs was determined with regard to serum IgG recognition and placental receptor binding. Discordance between the protease sensitivity for these phenomena was identified; this may have implications for PAM vaccine design. The completion of several Plasmodium genome sequences and advances in mass spectrometry have opened up the field of proteomics t...

The American journal of tropical medicine and hygiene, Jan 5, 2018

In 2004, there existed limited tuberculosis (TB) research capacity in the country of Georgia. In ... more In 2004, there existed limited tuberculosis (TB) research capacity in the country of Georgia. In response, a collaborative research training program (RTP) supported by a National Institutes of Health Fogarty International Center Global Infectious Diseases grant was formed between a U.S. academic institution and the National Center for Tuberculosis and Lung Disease (NCTLD) and other institutions in Georgia. We sought to assess outcomes of this RTP. The TB RTP combined didactic and mentored research training for Georgian trainees. Long-term trainees were supported for a 2-year period and with posttrainee career development mentoring. Metrics used to measure program performance included publications, grants received, and career advancement. From 2004 to 2015, 20 trainees participated in the program with 15 (75%) authoring a total of 65 publications in PubMed-listed journals. The median number of publications per trainee was six (interquartile range 2-14). A total of 16 (80%) trainees r...

Malaria journal, Jan 6, 2004

The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endo... more The ability of Plasmodium falciparum-infected erythrocytes to adhere to the microvasculature endothelium is thought to play a causal role in malaria pathogenesis. Cytoadhesion to endothelial receptors is generally found to be highly sensitive to trypsinization of the infected erythrocyte surface. However, several studies have found that parasite adhesion to placental receptors can be markedly less sensitive to trypsin. This study investigates whether chondroitin sulphate A (CSA) binding parasites express trypsin-resistant variant surface antigens (VSA) that bind female-specific antibodies induced as a result of pregnancy associated malaria (PAM). Fluorescence activated cell sorting (FACS) was used to measure the levels of adult Scottish and Ghanaian male, and Ghanaian pregnant female plasma immunoglobulin G (IgG) that bind to the surface of infected erythrocytes. P. falciparum clone FCR3 cultures were used to assay surface IgG binding before and after selection of the parasite for a...

Molecular Approaches toward Targeted Drug Development, 2011

... The vicious cycle of diarrhea and malnutrition imperils the health of infants and small child... more ... The vicious cycle of diarrhea and malnutrition imperils the health of infants and small children in the impoverished regions of the ... in mouse microsomes – a problem which must clearly be overcome if these compounds are to advance into a mouse model of cryptosporidiosis. ...

The Journal of Cell Biology, 2001

igh molecular weight homologues of gp91 phox, the superoxide-generating subunit of phagocyte nico... more igh molecular weight homologues of gp91 phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91 pho x domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. el-H egans . RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di-and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed crosslinking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix. *Abbreviations used in this paper: Ce-Duox, Caenorhabditis elegans Duox; dsRNA, double-stranded RNA; FAD, flavin adenine dinucleotide; h-Duox, human Duox; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate; RACE, rapid amplification of cDNA ends; RNAi, RNA interference; TMB, 3,3 Ј ,5,5 Ј -tetramethylbenzidine.

PLoS Neglected Tropical Diseases, 2010

Background: The protozoan parasite Cryptosporidium parvum is responsible for significant disease ... more Background: The protozoan parasite Cryptosporidium parvum is responsible for significant disease burden among children in developing countries. In addition Cryptosporidiosis can result in chronic and life-threatening enteritis in AIDS patients, and the currently available drugs lack efficacy in treating these severe conditions. The discovery and development of novel anti-cryptosporidial therapeutics has been hampered by the poor experimental tractability of this pathogen. While the genome sequencing effort has identified several intriguing new targets including a unique inosine monophosphate dehydrogenase (IMPDH), pursuing these targets and testing inhibitors has been frustratingly difficult.

Molecular and Cellular Biology, 2000

A large number of neuron-specific genes characterized to date are under the control of negative t... more A large number of neuron-specific genes characterized to date are under the control of negative transcriptional regulation. Many promoter regions of neuron-specific genes possess the repressor element repressor element 1/neuron-restrictive silencing element (RE1/NRSE). Its cognate binding protein, REST/NRSF, is an essential transcription factor; its null mutations result in embryonic lethality, and its dominant negative mutants produce aberrant expression of neuron-specific genes. REST/NRSF acts as a regulator of neuronspecific gene expression in both nonneuronal tissue and developing neurons. Here, we shown that heterologous expression of REST/NRSF in Saccharomyces cerevisiae is able to repress transcription from yeast promoters engineered to contain RE1/NRSEs. Moreover, we have taken advantage of this observation to show that this repression requires both yeast Sin3p and Rpd3p and that REST/NRSF physically interacts with the product of the yeast SIN3 gene in vivo. Furthermore, we show that REST/NRSF binds mammalian SIN3A and HDAC-2 and requires histone deacetylase activity to repress neuronal gene transcription in both nonneuronal and neuronal cell lines. We show that REST/NRSF binding to RE1/NRSE is accompanied by a decrease in the acetylation of histones around RE1/NRSE and that this decrease requires the N-terminal Sin3p binding domain of REST/NRSF. Taken together, these data suggest that REST/NRSF represses neuronal gene transcription by recruiting the SIN3/HDAC complex.

Molecular and Biochemical Parasitology, 2004

Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo... more Improved Plasmodium falciparum cDNA expression libraries were constructed by combining mRNA oligo-capping with in vitro recombination and directional cloning of cDNA inserts into a plasmid vector that expresses sequences as thioredoxin fusion proteins. A novel procedure has also been developed for the rapid identification of seropositive clones on high-density filters, using direct labelling of P. falciparum immune immunoglobulin with fluorescein isothiocynate (FITC). This approach combines the advantages of recombination-assisted cDNA cloning with high throughput, non-radioactive serological screening of expression libraries. Production of replicate colony matrices allows the identification of antigens recognised by different pools with different specificities from residents of a malaria endemic region. Analyses of DNA sequences derived from sero-reactive colonies indicate that this is an effective method for producing recombinant proteins that react with antibodies from malaria-exposed individuals. This approach permits the systematic construction of a database of antigenic proteins recognised by sera from malaria-exposed individuals.

Malaria Journal, 2007

Background: Sensitive detection of parasite surface antigens expressed on erythrocyte membranes i... more Background: Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites.

Life Sciences, 1999

The specific cellular response to muscarinic receptor activation is dependent upon appropriate ex... more The specific cellular response to muscarinic receptor activation is dependent upon appropriate expression of each of the five muscarinic receptor genes by individual cells.

Journal of Medicinal Chemistry, 2012

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine.... more Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5'-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high-throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC(50) < 2 nM) against CpIMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model.

Journal of Biological Chemistry, 2010

Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised... more Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k cat ‫؍‬ 0.28 s ؊1 and K m,ATP ‫؍‬ 140 M are similar to those of human TK1, the value of K m (thymidine) ‫؍‬ 48 M is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.

Chemistry & Biology, 2008

Cryptosporidium parvum is an important human pathogen and potential bio-terrorism agent. No vacci... more Cryptosporidium parvum is an important human pathogen and potential bio-terrorism agent. No vaccines exist against C. parvum, the drugs currently approved to treat cryptosporidiosis are ineffective, and drug discovery is challenging because the parasite cannot be maintained continuously in cell culture. Mining the sequence of the C. parvum genome has revealed that the only route to guanine nucleotides is via inosine-5′-monophosphate dehydrogenase (IMPDH). Moreover, phylogenetic analysis suggests that the IMPDH gene was obtained from bacteria by lateral gene transfer. Here we exploit the unexpected evolutionary divergence of parasite and host enzymes by designing a high throughput screen to target the most diverged portion of the IMPDH active site. We have identified four parasite-selective IMPDH inhibitors that display antiparasitic activity with greater potency than paromomycin, the current "gold standard" for anticryptosporidial activity.

Cellular Microbiology, 2007

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family... more The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a > 100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum bloodstage parasites and are exposed on the surface of haemolymph-and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

Bioorganic & Medicinal Chemistry Letters, 2012

Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. p... more Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. parvum and C. hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazolebased inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro.

International Journal of Women's Health, 2015