Keith M. Woods

Efficacy of 101 antimicrobials and other agents on the development of Cryptosporidium parvum in vitro

An in-situ ELISA was used as a primary screen to test the effects of 101 antimicrobials and other agents on the development of Cryptosporidium parvum in vitro. Over 40 of the compounds displayed some form of anticryptosporidial activity, and dose-response curves were generated for 40 of these. The in-situ ELISA makes a highly effective primary, pharmaceutical screen for C parvum, to be used prior to more detailed microscopical, toxicological or in-vivo assays.

Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum

We report here the molecular analysis of a Type I fatty acid synthase in the apicomplexan Cryptosporidium parvum (CpFAS1). The CpFAS1 gene encodes a multifunctional polypeptide of 8243 amino acids that contains 21 enzymatic domains. This CpFAS1 structure is distinct from that of mammalian Type I FAS, which contains only seven enzymatic domains. The CpFAS1 domains are organized into: (i) a starter unit consisting of a fatty acid ligase and an acyl carrier protein; (ii) three modules, each containing a complete set of six enzymes (acyl transferase, ketoacyl synthase, ketoacyl reductase, dehydrase, enoyl reductase, and acyl carrier protein) for the elongation of fatty acid C2-units; and (iii) a terminating domain whose function is as yet unknown. The CpFAS1 gene is expressed throughout the life cycle of C. parvum, since its transcripts and protein were detected by RT-PCR and immunofluorescent localization, respectively. This cytosolic Type I CpFAS1 differs from the organellar Type II FAS enzymes identified from Toxoplasma gondii and Plasmodium falciparum which are targetted to the apicoplast, and are sensitive to inhibition by thiolactomycin. That the discovery of CpFAS1 may provide a new biosynthetic pathway for drug development against cryptosporidiosis, is indicated by the efficacy of the FAS inhibitor cerulenin on the growth of C. parvum in vitro.

Virus‐like, double‐stranded RNAs in the parasitic protozoan Cryptosporidium parvum

We have discovered and analysed two novel, linear extrachromosomal double‐stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L‐dsRNA) had homology with viral RNA‐dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S‐dsRNA) had limited similarity with mitogen‐activated c‐June NH2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus‐like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.

Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy

This study demonstrates that polyamine biosynthesis in Cryptosporidium parvum occurs via a pathway chiefly found in plants and some bacteria. The lead enzyme of this pathway, arginine decarboxylase (ADC) was sensitive to the specific, irreversible inhibitor DL-alpha-difluoromethyl-arginine (IC50 30 microM), and intracellular growth of C. parvum was significantly reduced by inhibitors of ADC. No activity was detected using ornithine as substrate, and the irreversible inhibitor of ornithine decarboxylase, DL-alpha-difluoromethyl-ornithine, had no effect upon ADC activity or upon growth of the parasite. Back-conversion of spermine to spermidine and putrescine via spermidine:spermine-N1-acetyltransferase (SSAT) was also detected. Compounds such as his(ethyl)norspermine, which have been demonstrated to down-regulate SSAT activity in tumor cells, were synergistic in the inhibition of growth when used in combination with inhibitors of the forward pathway. Thus, C. parvum differs fundamentally in its polyamine metabolism from the majority of eukaryotes, including humans. Such differences indicate that polyamine metabolism may serve as a chemotherapeutic target in this organism.

Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen Cryptosporidium parvum In Vitro

ABSTRACT We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (CT[P18S]) and host cells (CT[H18S]), and evaluates the relative expression between parasite and host rRNA levels (i.e., ΔCT = CT[P18S] − CT[H18S]) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (ΔΔCT) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in ∼3 h) in dead parasites is faster than that of DNA (99% in ∼24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 μg/ml and 89.7 to 119.0 μg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets.

Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell

The ability of membrane antigens on sporozoites of the intestinal pathogen, Cryptosporidium parvum, to bind host cell surface antigens was investigated. A novel membrane-associated protein of approximately 47 kDa, designated CP47, was found to possess significant binding affinity for the surface of both human and animal ileal cells. This protein was purified by a combination of anion-exchange chromatography on FPLC and immunoaffinity chromatography. Purified CP47 demonstrated competitive binding with parasite-associated membrane antigens to membranes of HCT-8 and ileal cells in a dose-dependent manner. Furthermore, the binding activity of CP47 was found to be Mn2+-sensitive, and was completely inhibited in the presence of 10 mM MnCl2. These results were consistent with earlier findings demonstrating the inhibitory effect of Mn2+ ions on Cryptosporidium infection both in vitro and in vivo (Nesterenko et al., Biol. Trace Elem. Res. 56 (1997) 243-253). Immunoelectron microscopy using gold-conjugated antibodies revealed CP47 to be localized at the apical end of the sporozoites. A single protein with an electrophoretic mobility of 57 kDa was purified from host cell membranes using CP47-Affigel. Similarly, affinity purification of this protein was abrogated in the presence of Mn2+. These data suggest that a novel parasite protein, CP47, may play an important role in sporozoite/host cell attachment.

Irreversible UV Inactivation of Cryptosporidium spp. Despite the Presence of UV Repair Genes1

Abstract Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm2 resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92–98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

Three distinct cell phenotypes of induced-TNF cytotoxicity and their relationship to apoptosis.

We have identified three distinct cell phenotypes with respect to the conditions under which cells became susceptible to TNF‐mediated lysis. These conditions include: 1) treatment with the protein synthesis inhibitor, cycloheximide; 2) contact with activated macrophages, and 3) infection with vaccinia virus. Whereas vaccinia virus‐infected 3T3 cells became sensitive to soluble TNF, F5b cells required contact with activated macrophages. We showed that the “macrophage‐resistant” F5m cells did not become sensitive to TNF or to killing by activated macrophages after infection with vaccinia virus. Therefore, vaccinia infection does not sensitize all cells to TNF. We also determined the pathways of lysis for cells after sensitization. Whereas 3T3, LM929, and F5b cells were killed by the process of necrosis, F5m cells lysis was characterized by the release of low mol wt DNA fragments (apoptosis).

Cryspovirus: a new genus of protozoan viruses in the family Partitiviridae

The family Partitiviridae includes plant and fungal viruses with bisegmented dsRNA genomes and isometric virions in which the two genome segments are packaged separately and used as templates for semiconservative transcription by the viral polymerase. A new genus, Cryspovirus, has been approved for this family. Its name is based on that of the host genus, Cryptosporidium, which encompasses several species of apicomplexan parasites that infect a wide range of mammals, birds, and reptiles, and are a major cause of human diarrheal illness worldwide. The type species of the new genus is Cryptosporidium parvum virus 1. Distinguishing characteristics include infection of a protozoan host, a smaller capsid protein than found in other members of the family Partitiviridae, and sequence-based phylogenetic divergence.

Domain V Peptides Inhibit β2-Glycoprotein I-Mediated Mesenteric Ischemia/Reperfusion-Induced Tissue Damage and Inflammation

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti–β2-GPI Abs into Rag-1−/− mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.