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Karen S. Anderson - One of the best experts on this subject based on the ideXlab platform.
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targeting the ts dimer interface in bifunctional Cryptosporidium hominis ts dhfr from parasitic protozoa virtual screening identifies novel ts allosteric inhibitors
Bioorganic & Medicinal Chemistry Letters, 2020Co-Authors: Victor G Ruiz, Vidya Prasanna Kumar, William L. Jorgensen, Daniel J Czyzyk, Karen S. AndersonAbstract:Abstract Effective therapies are lacking to treat gastrointestinal infections caused by the genus Cryptosporidium, which can be fatal in the immunocompromised. One target of interest is Cryptosporidium hominis (C. hominis) thymidylate synthase-dihydrofolate reductase (ChTS-DHFR), a bifunctional enzyme necessary for DNA biosynthesis. Targeting the TS-TS dimer interface is a novel strategy previously used to identify inhibitors against the related bifunctional enzyme in Toxoplasma gondii. In the present study, we target the ChTS dimer interface through homology modeling and high-throughput virtual screening to identifying allosteric, ChTS-specific inhibitors. Our work led to the discovery of methylenedioxyphenyl-aminophenoxypropanol analogues which inhibit ChTS activity in a manner that is both dose-dependent and influenced by the conformation of the enzyme. Preliminary results presented here include an analysis of structure activity relationships and a ChTS-apo crystal structure of ChTS-DHFR supporting the continued development of inhibitors that stabilize a novel pocket formed in the open conformation of ChTS-TS.
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novel allosteric covalent inhibitors of bifunctional Cryptosporidium hominis ts dhfr from parasitic protozoa identified by virtual screening
Bioorganic & Medicinal Chemistry Letters, 2019Co-Authors: Victor G Ruiz, William L. Jorgensen, Daniel J Czyzyk, M Valhondo, Karen S. AndersonAbstract:Abstract Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region “crossover helix” to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the non-active site pocket.
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Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis Thymidylate Synthase-Dihydrofolate Reductase
Bioorganic & medicinal chemistry letters, 2014Co-Authors: Vidya Prasanna Kumar, Kathleen M. Frey, Yiqiang Wang, Aleem Gangjee, José A. Cisneros, Alejandro Castellanos-gonzalez, A. Clinton White, William L. Jorgensen, Karen S. AndersonAbstract:Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.
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Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors.
Bioorganic & medicinal chemistry letters, 2013Co-Authors: Vidya Prasanna Kumar, Kathleen M. Frey, Yiqiang Wang, Hitesh K. Jain, Aleem Gangjee, Karen S. AndersonAbstract:Abstract Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals. 1,2 Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3- d ]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase ( Ch TS) inhibitors. Crystal structure in complex with the most potent compound, a 2′-chlorophenyl with a sulfur bridge with a K i of 8.83 ± 0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop Ch TS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of Ch TS specific inhibitors.
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Disruption of the Crossover Helix Impairs Dihydrofolate Reductase Activity in the Bifunctional Enzyme TS-DHFR from Cryptosporidium hominis
The Biochemical journal, 2009Co-Authors: Melissa A. Vargo, W. Edward Martucci, Karen S. AndersonAbstract:In contrast to most species, including humans, having monofunctional forms of the folate biosynthetic enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR), several pathogenic protozoal parasites, including Cryptosporidium hominus, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) Cryptosporidium hominis reveals a dimer with a “crossover helix”, a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In this study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an “alanine face” enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit are mutated to alanine, a “glycine face” enzyme in which the same residues are mutated to glycine, and an “all alanine” helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations cause a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine face mutant enzyme is 30 s-1, the glycine face mutant enzyme is 17 s-1, and the all alanine helix enzyme is 16 s-1, all substantially impaired from the wild-type DHFR activity of 152 s-1. It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS-DHFR that could be exploited as a target for species-specific non-active site inhibitors.
Amy C. Anderson - One of the best experts on this subject based on the ideXlab platform.
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Highly efficient ligands for dihydrofolate reductase from Cryptosporidium hominis and Toxoplasma gondii inspired by structural analysis.
Journal of medicinal chemistry, 2007Co-Authors: Phillip M. Pelphrey, Veljko M. Popov, Yale A. Fillingham, Dennis L. Wright, Tammy M. Joska, Jennifer M. Beierlein, Erin S. D. Bolstad, Amy C. AndersonAbstract:The search for effective therapeutics for cryptosporidiosis and toxoplasmosis has led to the discovery of novel inhibitors of dihydrofolate reductase (DHFR) that possess high ligand efficiency: compounds with high potency and low molecular weight. Detailed analysis of the crystal structure of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis and a homology model of DHFR from Toxoplasma gondii inspired the synthesis of a new series of compounds with a propargyl-based linker between a substituted 2,4-diaminopyrimidine and a trimethoxyphenyl ring. An enantiomerically pure compound in this series exhibits IC50 values of 38 and 1 nM against C. hominis and T. gondii DHFR, respectively. Improvements of 368-fold or 5714-fold (C. hominis and T. gondii) relative to trimethoprim were generated by synthesizing just 14 new analogues and by adding only a total of 52 Da to the mass of the parent compound, creating an efficient ligand as an excellent candidate for further study.
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Analysis of complexes of inhibitors with Cryptosporidium hominis DHFR leads to a new trimethoprim derivative.
Bioorganic & medicinal chemistry letters, 2006Co-Authors: Veljko M. Popov, David C. Chan, Yale A. Fillingham, W. Atom Yee, Dennis L. Wright, Amy C. AndersonAbstract:Abstract Cryptosporidiosis, an opportunistic infection affecting immunocompromised patients, the elderly, and children, is still an untreatable disease since the causative agent, Cryptosporidium hominis, is essentially resistant to all clinically used antimicrobial agents. In order to accelerate the design of new potent and selective inhibitors targeting dihydrofolate reductase of C. hominis (ChDHFR), we determined the structural basis for the potency of existing DHFR inhibitors using superpositions of the structure of ChDHFR with other species and analysis of active site complexes of ChDHFR bound to ligands exhibiting a wide range of IC50 values. This information was used to develop an accurate docking model capable of identifying potent inhibitors in silico. A series of C7-trimethoprim derivatives, designed to exploit a unique pocket in ChDHFR, was synthesized and evaluated; 7-ethyl TMP has four times higher activity than TMP against ChDHFR.
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Two crystal structures of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis reveal protein-ligand interactions including a structural basis for observed antifolate resistance.
Acta crystallographica. Section F Structural biology and crystallization communications, 2005Co-Authors: Amy C. AndersonAbstract:Cryptosporidium hominis is a protozoan parasite that causes acute gastrointestinal illness. There are no effective therapies for cryptosporidiosis, highlighting the need for new drug-lead discovery. An analysis of the protein-ligand interactions in two crystal structures of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from C. hominis, determined at 2.8 and 2.87 A resolution, reveals that the interactions of residues Ile29, Thr58 and Cys113 in the active site of C. hominis DHFR provide a possible structural basis for the observed antifolate resistance. A comparison with the structure of human DHFR reveals active-site differences that may be exploited for the design of species-selective inhibitors.
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The crystal structure of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis reveals a novel architecture for the bifunctional enzyme.
Journal of Eukaryotic Microbiology, 2003Co-Authors: Robert H. O'neil, Ryan H. Lilien, Bruce R. Donald, Robert M. Stroud, Amy C. AndersonAbstract:Cryptosporidium hominis is an emerging pathogen that primarily affects immune-compromised patients, including those with AIDS. There is no effective cure at this point for the degenerative wasting disease that can follow infection. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a bifunctional enzyme in C. hominis and is crucial in the folate cycle for the production of dTMP, one of the four DNA bases. DHFR-TS is an excellent drug target since its inhibition leads to the death of the pathogen. In apicomplexan protozoa, including C. hominis, the gene for DHFR-TS can be partitioned into a DHFR domain, a linker domain and a TS domain. In human cells, DHFR and TS are separate, monofunctional enzymes. We have determined the X-ray crystal structure of DHFR-TS from C. hominis. The structure reveals that the linker polypeptide between the DHFR and TS domains has important structural interactions with the opposite monomer of the homodimeric enzyme. A comparison of the structure of DHFR-TS from C. hominis and structures of DHFR-TS from Plasmodium falciparum [11] and Leishmania major [5] shows that there are significant structural differences between the apicomplexan and kinetoplastid forms of the enzyme. Additionally, the DHFR and TS domains of the C. hominis enzyme have important sequence and structural differences from the human forms of the enzymes. The species-specific differences are important for future inhibitor design targeting C. hominis.
William L. Jorgensen - One of the best experts on this subject based on the ideXlab platform.
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targeting the ts dimer interface in bifunctional Cryptosporidium hominis ts dhfr from parasitic protozoa virtual screening identifies novel ts allosteric inhibitors
Bioorganic & Medicinal Chemistry Letters, 2020Co-Authors: Victor G Ruiz, Vidya Prasanna Kumar, William L. Jorgensen, Daniel J Czyzyk, Karen S. AndersonAbstract:Abstract Effective therapies are lacking to treat gastrointestinal infections caused by the genus Cryptosporidium, which can be fatal in the immunocompromised. One target of interest is Cryptosporidium hominis (C. hominis) thymidylate synthase-dihydrofolate reductase (ChTS-DHFR), a bifunctional enzyme necessary for DNA biosynthesis. Targeting the TS-TS dimer interface is a novel strategy previously used to identify inhibitors against the related bifunctional enzyme in Toxoplasma gondii. In the present study, we target the ChTS dimer interface through homology modeling and high-throughput virtual screening to identifying allosteric, ChTS-specific inhibitors. Our work led to the discovery of methylenedioxyphenyl-aminophenoxypropanol analogues which inhibit ChTS activity in a manner that is both dose-dependent and influenced by the conformation of the enzyme. Preliminary results presented here include an analysis of structure activity relationships and a ChTS-apo crystal structure of ChTS-DHFR supporting the continued development of inhibitors that stabilize a novel pocket formed in the open conformation of ChTS-TS.
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novel allosteric covalent inhibitors of bifunctional Cryptosporidium hominis ts dhfr from parasitic protozoa identified by virtual screening
Bioorganic & Medicinal Chemistry Letters, 2019Co-Authors: Victor G Ruiz, William L. Jorgensen, Daniel J Czyzyk, M Valhondo, Karen S. AndersonAbstract:Abstract Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region “crossover helix” to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the non-active site pocket.
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Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis Thymidylate Synthase-Dihydrofolate Reductase
Bioorganic & medicinal chemistry letters, 2014Co-Authors: Vidya Prasanna Kumar, Kathleen M. Frey, Yiqiang Wang, Aleem Gangjee, José A. Cisneros, Alejandro Castellanos-gonzalez, A. Clinton White, William L. Jorgensen, Karen S. AndersonAbstract:Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.
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Novel non-active site inhibitor of Cryptosporidium hominis TS-DHFR identified by a virtual screen
Bioorganic & medicinal chemistry letters, 2008Co-Authors: W. Edward Martucci, William L. Jorgensen, Marina Udier-blagovic, Chloé E. Atreya, Oladapo Babatunde, Melissa A. Vargo, Karen S. AndersonAbstract:The essential enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a validated drug target for many pathogens, but has been elusive in Cryptosporidium hominis, as active site inhibitors of the enzymes from related parasitic protozoa show decreased potency and lack of species specificity over the human enzymes. As a rational approach to discover novel inhibitors, we conducted a virtual screen of a non-active site pocket in the DHFR linker region. From this screen, we have identified and characterized a noncompetitive inhibitor, flavin mononucleotide (FMN), with micromolar potency that is selective for ChTS-DHFR versus the human enzymes. These results describe a novel allosteric pocket amenable to inhibitor targeting, and a lead compound with which to move towards potent, selective inhibitors of ChTS-DHFR.
Lihua Xiao - One of the best experts on this subject based on the ideXlab platform.
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Cryptosporidium parvum and Cryptosporidium hominis subtypes in crab-eating macaques.
Parasites & vectors, 2019Co-Authors: Li Chen, Yaoyu Feng, Wen Jiang, Jianguo Zhao, Yaqiong Guo, Chenghong Liao, Qian Han, Lihua XiaoAbstract:Background Non-human primates are often infected with human-pathogenic Cryptosporidium hominis subtypes, but rarely with Cryptosporidium parvum. In this study, 1452 fecal specimens were collected from farmed crab-eating macaques (Macaca fascicularis) in Hainan, China during the period April 2016 to January 2018. These specimens were analyzed for Cryptosporidium species and subtypes by using PCR and sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively.
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Persistent Occurrence of Cryptosporidium hominis and Giardia duodenalis Subtypes in a Welfare Institute
Frontiers Media S.A., 2018Co-Authors: Yuanfei Wang, Yaoyu Feng, Yaqiong Guo, Lin Wang, Rui Wang, Lihua XiaoAbstract:Few data are available on the transmission dynamics of intestinal protozoa in children in welfare institutes. In this study, fecal specimens were collected from 396 children in a welfare institute in Shanghai, China during December 2011 (207 specimens), June 2012 (78 specimens), and September 2013 (111 specimens), and examined for Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by PCR analysis of the small subunit rRNA, triosephosphate isomerase, and internal transcribed spacer genes, respectively. The Cryptosporidium hominis and G. duodenalis assemblage A identified were further subtyped by multilocus sequence typing. Altogether, Cryptosporidium was detected in 39 (9.8%) children, with infection rates of 11.6% (24/207), 9.0% (7/78), and 7.2% (8/111) in December 2011, June 2012, and September 2013, respectively. Infection rates were higher in children of 0–12 months (20.4% compared to 0–7.3% in other age groups, P = 0.0001) and those with diarrhea (17.9% compared to 7.7% in those with no diarrhea, P = 0.006). In contrast, G. duodenalis was detected in 161/396 (40.7%), with infection rates of 48.3% (100/207), 35.9% (28/78), and 29.7% (33/111) in December 2011, June 2012, and September 2013, respectively. There were no significant gender- or diarrhea-associated differences, but the G. duodenalis infection rate in children of 13–24 months (50%) was significantly higher than in the age groups of 0–12 months and > 48 months (29.8–36.5%, P = 0.021). Co-infection of Cryptosporidium and G. duodenalis was seen in 19 (4.8%) children, but no E. bieneusi infection was detected in this study. All Cryptosporidium-positive specimens belonged to the subtype IaA14R4 of C. hominis, while all G. duodenalis-positive specimens belonged to sub-assemblage AII. Both were the same subtypes in a previous outbreak of cryptosporidiosis and giardiasis in a hospital ward hosting children from the welfare institute. Results of the study indicate that there was a persistent occurrence of limited C. hominis and G. duodenalis subtypes in the small enclosed community, with differences in age distribution and association with diarrhea occurrence between cryptosporidiosis and giardiasis
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Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum.
BMC genomics, 2015Co-Authors: Yaqiong Guo, Yaoyu Feng, Kevin Tang, Lori A. Rowe, Dawn M. Roellig, Kristine Knipe, Michael Frace, Chunfu Yang, Lihua XiaoAbstract:Background Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis–associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome.
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Microsporidia and Cryptosporidium in horses and donkeys in Algeria: detection of a novel Cryptosporidium hominis subtype family (Ik) in a horse.
Veterinary Parasitology, 2015Co-Authors: Abd Elkarim Laatamna, Lihua Xiao, Pavla Wagnerová, Bohumil Sak, Dana Květoňová, Michael Rost, John Mcevoy, Ahmed Rachid Saadi, Meriem Aissi, Martin KváčAbstract:Abstract A total of 219 and 124 individual fecal samples of horses and donkeys, respectively, were screened for the presence of Cryptosporidium spp., Encephalitozoon spp., and Enterocytozoon bieneusi DNA by genus-specific nested PCR. Isolates were genotyped by sequence analysis of SSU rRNA, GP60, TRAP-C1, COWP, and HSP70 loci in Cryptosporidium , and the ITS region in microsporidia. Cryptosporidium spp. was detected on 3/18 horse farms and 1/15 farms where donkeys were kept. Overall, five (2.3%) horse and two (1.6%) donkey specimens were PCR positive for Cryptosporidium . Genotyping at SSU and GP60 loci revealed that three isolates from horses and donkeys were C. parvum subtype family IIaA16G1R1, one isolate from a horse was, C. muris RN66, and one isolate from a donkey was C. muris TS03. An isolate from a horse shared 99.4% and 99.3% similarity with Cryptosporidium hominis and C. cuniculus , respectively, at the SSU locus. This isolate shared 100% identity with C. hominis at the TRAP-C1, COWP, and HSP70 loci, and it was from the novel gp60 subtype family IkA15G1. Microsporidia were found on 6/18 horse and 2/15 donkey farms. E. bieneusi was identified in 6.8% (15/219) and 1.6% (2/124), and Encephalitozoon cuniculi was identified in 1.8% (4/219) and 1.6% (2/124), of horses and donkeys, respectively. Three genotypes of E. cuniculi (I, II and III) were detected in horses, and E. cuniculi genotype II was detected in donkeys. Four genotypes of E. bieneusi (horse1, horse 2, CZ3, D) were described in horses. An additional five horses and two donkeys were positive for E. bieneusi , but the isolated were not genotyped. Neither Cryptosporidium nor microsporidia prevalence were affected by sex, age, type of breeding, or whether the host was a horse or a donkey.
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Multilocus Sequence Typing of an Emerging Cryptosporidium hominis Subtype in the United States
Journal of clinical microbiology, 2013Co-Authors: Yaoyu Feng, Michele C. Hlavsa, Narry Tiao, Lihua XiaoAbstract:ABSTRACT The United States has experienced a substantial increase in the reported incidence of cryptosporidiosis since 2005. Accompanying this is the emergence of a new subtype of Cryptosporidium hominis based on variation at the 60-kDa glycoprotein (gp60) locus, IaA28R4, which has become a frequently identified subtype in both sporadic and outbreak-related cases. In this study, using multilocus sequence typing (MLST) at eight genetic loci, we characterized 62 specimens of IaA28R4 and 33 specimens of three other gp60 subtypes of C. hominis from four U.S. states with increased cryptosporidiosis incidences during the summer of 2008. Extensive genetic heterogeneity was seen within the gp60 subtype IaA28R4, but specimens from Ohio and southwestern states formed two distinct subpopulations, suggesting that there were at least two origins of IaA28R4 within the United States. Discordance in typing results was observed between gp60 and other genetic markers, especially DZ-HRGP, and this discordance was largely the result of genetic recombination within the gp60 subtype IaA28R4. The results of population genetic analyses supported the presence of two subpopulations of IaA28R4 and the occurrence of genetic recombination within this gp60 subtype. Thus, the IaA28R4 subtype at gp60 is likely a fitness marker for C. hominis, and genetic recombination is potentially a driving force in the emergence of the virulent IaA28R4 subtype in the United States. A rapid evolution of IaA28R4 was indicated by the observation of multiple MLST subtypes of IaA28R4 within two large outbreaks that lasted for extended periods and involved multiple swimming pools.
Robin B. Gasser - One of the best experts on this subject based on the ideXlab platform.
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high resolution melting curve hrm analysis for the diagnosis of cryptosporidiosis in humans
Molecular and Cellular Probes, 2009Co-Authors: Aradhana Pangasa, Aaron R. Jex, Bronwyn E Campbell, Nathan J Bott, Margaret Whipp, Geoff Hogg, Melita A Stevens, Robin B. GasserAbstract:Cryptosporidiosis of humans is an intestinal disease caused predominantly by infection with Cryptosporidium hominis or C. parvum. This disease is transmitted mainly via the faecal-oral route (water or food) and has major socioeconomic impact globally. The diagnosis and genetic characterization of the main species and population variants (also called ‘‘genotypes’’ and ‘‘subgenotypes’’) of Cryptosporidium infecting humans is central to the prevention, surveillance and control of cryptosporidiosis, particularly as there is presently no cost effective anti-cryptosporidial chemotherapeutic regimen or vaccine available. In the present study, we established a polymerase chain reaction (PCR)-coupled high resolution melting-curve (HRM) analysis method, utilizing the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker, for the diagnosis of Cryptosporidium hominis, C. parvum or C. meleagridis infection. An evaluation of the method revealed intra- and inter-assay variabilities of <1.5 and 3.5%, respectively. Cryptosporidium hominis, C. parvum and C. meleagridis were detected in 97, 44 and 2, respectively, of the 143 Cryptosporidium oocyst DNA samples originating from Australians with clinical cryptosporidiosis. The melting profiles characterized by peaks of 72.47 � 0.33
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High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans.
Molecular and cellular probes, 2008Co-Authors: Aradhana Pangasa, Aaron R. Jex, Bronwyn E Campbell, Nathan J Bott, Margaret Whipp, Geoff Hogg, Melita A Stevens, Robin B. GasserAbstract:Cryptosporidiosis of humans is an intestinal disease caused predominantly by infection with Cryptosporidium hominis or C. parvum. This disease is transmitted mainly via the faecal-oral route (water or food) and has major socioeconomic impact globally. The diagnosis and genetic characterization of the main species and population variants (also called ‘‘genotypes’’ and ‘‘subgenotypes’’) of Cryptosporidium infecting humans is central to the prevention, surveillance and control of cryptosporidiosis, particularly as there is presently no cost effective anti-cryptosporidial chemotherapeutic regimen or vaccine available. In the present study, we established a polymerase chain reaction (PCR)-coupled high resolution melting-curve (HRM) analysis method, utilizing the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker, for the diagnosis of Cryptosporidium hominis, C. parvum or C. meleagridis infection. An evaluation of the method revealed intra- and inter-assay variabilities of
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Analysis of the genetic diversity within Cryptosporidium hominis and Cryptosporidium parvum from imported and autochtonous cases of human cryptosporidiosis by mutation scanning.
Electrophoresis, 2008Co-Authors: Aaron R. Jex, Robin B. GasserAbstract:The present study investigated sequence variation in part of the 60 kilodalton glycoprotein (pgp60) gene among Cryptosporidium hominis and Cryptosporidium parvum isolates (n = 115) from citizens of the UK inferred to have been infected whilst travelling abroad (to 25 countries) or in the UK. The genomic DNA samples from these isolates were subjected to PCR-coupled single-strand conformation polymorphism analysis, followed by targeted sequencing of pgp60. Individual samples were classified to the genotypic and subgenotypic levels based on phylogenetic analysis (Bayesian inference) of pgp60 data, including published sequences for comparison. Based on this analysis, five C. hominis (Ia-If) and four C. parvum (IIa, IIc-IIe) genotypes were identified, equating to 16 and 10 subgenotypes, respectively. Of these genotypes, C. hominis Ib was predominant (n = 82). Interestingly, one subgenotype (C. hominis Ib A10G2R2) accounted for the majority of the samples examined and was identified in travellers to 14 countries; the examination of published records suggested that C. hominis Ib A10G2R2 has a global distribution. Numerous new and seemingly rare subgenotypes (eight for C. hominis and six for C. parvum) were also discovered. In conclusion, the present study revealed substantial genetic variation in pgp60 within both C. hominis and C. parvum and emphasizes the need to undertake investigations of human and animal populations in countries for which there is no information on the genetic make-up of Cryptosporidium infecting humans.
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Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species.
International journal for parasitology, 2005Co-Authors: Rachel M. Chalmers, Lihua Xiao, Stephen J. Hadfield, Kristin Elwin, Simone M. Cacciò, Christobel Ferguson, Robin B. Gasser, Youssef G. Abs El-osta, Leo Heijnen, Martha Irvine SinclairAbstract:Abstract A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces ( n =54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis , SSCP analysis of ITS-2 was considered to have superior utility and determined ‘subgenotypes’ in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and ‘gp15’ provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium . This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.