Guan Zhu

A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium

Cryptosporidium spp are well recognised as causes of diarrhoeal disease during waterborne epidemics and in immunocompromised hosts. Studies have also drawn attention to an underestimated global burden and suggest major gaps in optimum diagnosis, treatment, and immunisation. Cryptosporidiosis is increasingly identified as an important cause of morbidity and mortality worldwide. Studies in low-resource settings and high-income countries have confirmed the importance of cryptosporidium as a cause of diarrhoea and childhood malnutrition. Diagnostic tests for cryptosporidium infection are suboptimum, necessitating specialised tests that are often insensitive. Antigen-detection and PCR improve sensitivity, and multiplexed antigen detection and molecular assays are underused. Therapy has some effect in healthy hosts and no proven efficacy in patients with AIDS. Use of cryptosporidium genomes has helped to identify promising therapeutic targets, and drugs are in development, but methods to assess the efficacy in vitro and in animals are not well standardised. Partial immunity after exposure suggests the potential for successful vaccines, and several are in development; however, surrogates of protection are not well defined. Improved methods for propagation and genetic manipulation of the organism would be significant advances.

Cryptosporidium and Cryptosporidiosis

Cryptosporidium is one of the most common enteric protozoan parasites of vertebrates with a wide host range that includes humans and domestic animals. It is a significant cause of diarrhoeal disease and an ubiquitous contaminant of water which serves as an excellent vehicle for transmission. A better understanding of the development and life cycle of Cryptosporidium, and new insights into its phylogenetic relationships, have illustrated the need to re-evaluate many aspects of the biology of Cryptosporidium. This has been reinforced by information obtained from the recent successful Cryptosporidium genome sequencing project, which has emphasised the uniqueness of this organism in terms of its parasite life style and evolutionary biology. This chapter provides an up to date review of the biology, biochemistry and host parasite relationships of Cryptosporidium.

What is the phylogenetic position of Cryptosporidium

The phylogenetic position of Cryptosporidium is elusive. Although previous studies based solely upon small-subunit (SSU) rRNA sequences suggested that the genus was an early emerging lineage among the Apicomplexa, bootstrap support for this placement was low. Here, the phylogenetic position of Cryptosporidium has been re-evaluated for SSU rRNA, fused SSU/large-subunit (LSU) rRNA and six protein sequences using traditional distance-based neighbour-joining, maximum-parsimony and maximum-likelihood methods of phylogenetic reconstruction as well as the new Slow-Fast analysis, which focuses upon the slowly evolving positions within sequences and is especially useful if a long-branch attraction (LBA) artefact is suspected. All the methods of reconstruction indicated a trend for the early emergence of Cryptosporidium at the base of the Apicomplexa and showed that an LBA artefact plays no role in this placement. Although the inclusion of additional numbers of neither species nor genes has significantly enhanced the bootstrap support for this phylogenetic position, recent biochemical, molecular and ultrastructural data are congruent with it. Therefore, we favour a working hypothesis that this genus constitutes an early emerging branch of the Apicomplexa.

A Genome-Sequence Survey for Ascogregarina taiwanensis Supports Evolutionary Affiliation but Metabolic Diversity between a Gregarine and Cryptosporidium

We have performed a whole-genome-sequence survey for the gregarine, Ascogregarina taiwanensis and herein describe both features unique to this early diverging apicomplexan and properties that unite it with Cryptosporidium, the Coccidia, and the Apicomplexa. Phylogenetic trees inferred from a concatenated protein sequence comprised of 10,750 amino acid positions, as well as the large subunit rRNA genes, robustly support phylogenetic affinity of Ascogregarina with Cryptosporidium at the base of the apicomplexan clade. Unlike Cryptosporidium, Ascogregarina possesses numerous mitochondrion-associated pathways and proteins, including enzymes within the Krebs cycle and a cytochrome-based respiratory chain. Ascogregarina further differs in the capacity for de novo synthesis of pyrimidines and amino acids. Ascogregarina shares with Cryptosporidium a Type I fatty acid synthase and likely a polyketide synthase. Cryptosporidium and Ascogregarina possess a large repertoire of multidomain surface proteins that align it with Toxoplasma and are proposed to be involved in coccidian-like functions. Four families of retrotransposable elements were identified, and thus, retroelements are present in Ascogregarina and Eimeria but not in other apicomplexans that have been analyzed. The sum observations suggest that Ascogregarina and Cryptosporidium share numerous molecular similarities, not only including coccidian-like features to the exclusion of Haemosporidia and Piroplasmida but also differ from each other significantly in their metabolic capacity.

Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.

Apical Organelle Discharge by Cryptosporidium parvum Is Temperature, Cytoskeleton, and Intracellular Calcium Dependent and Required for Host Cell Invasion

ABSTRACT The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasites intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.

Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src

BACKGROUND & AIMS Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia. METHODS In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion. RESULTS c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum -induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion. CONCLUSIONS C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.

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.

Current Progress in the Fatty Acid Metabolism in Cryptosporidium parvum1

Abstract Cryptosporidium parvum is one of the apicomplexans that can cause severe diarrhea in humans and animals. The slow development of anti-cryptosporidiosis chemotherapy is primarily due to the poor understanding on the basic metabolic pathways in this parasite. Many well-defined or promising drug targets found in other apicomplexans are either absent or highly divergent in C. parvum. The recently discovered apicoplast and its associated Type II fatty acid synthetic enzymes in Plasmodium, Toxoplasma, and Eimeria apicomplexans are absent in C. parvum, suggesting this parasite is unable to synthesize fatty acids de novo. However, C. parvum possesses a giant Type I fatty acid synthase (CpFAS1) that makes very long chain fatty acids using mediate or long chain fatty acids as precursors. Cryptosporidium also contains a Type I polyketide synthase (CpPKS1) that is probably involved in the production of unknown polyketide(s) from a fatty acid precursor. In addition to CpFAS1 and CpPKS1, a number of other enzymes involved in fatty acid metabolism have also been identified. These include a long chain fatty acyl elongase (LCE), a cytosolic acetyl-CoA carboxylase (ACCase), three acyl-CoA synthases (ACS), and an unusual “long-type” acyl-CoA binding protein (ACBP), which allows us to hypothetically reconstruct the highly streamlined fatty acid metabolism in this parasite. However, C. parvum lacks enzymes for the oxidation of fatty acids, indicating that fatty acids are not an energy source for this parasite. Since fatty acids are essential components of all biomembranes, molecular and functional studies on these critical enzymes would not only deepen our understanding on the basic metabolism in the parasites, but also point new directions for the drug discovery against C. parvum and other apicomplexan-based diseases.

Characterization in vitro and in vivo of resistance to ionophores in a strain of Eimeria tenella

A field isolate of Eimeria tenella (FS139) was propagated several times in chickens medicated with 200 ppm of dietary monensin. In a laboratory test with 2-wk-old-chickens, the strain was resistant to monensin, salinomycin, and lasalocid given at double use level and was resistant to narasin and maduramicin at the normal use level. In comparison, a laboratory strain (WIS) was controlled by the normal use level of each product. When free WIS sporozoites were treated in vitro with 1.0 microgram/ml of monensin for 0.5 or 4.0 hr at 41 C and inoculated into primary cultures of chicken kidney cells the invasion was reduced by 35.6% or 96.3%, but invasion of FS139 sporozoites was increased by 18.5% by 0.5 hr treatment and was about the same as controls after 2 hr of treatment. Few sporozoites from the WIS strain developed into schizonts, but numerous sporozoites from the FS139 strain developed into normal first and second generation schizonts. The structure of free WIS sporozoites was distorted after 3 hr of treatment with 2.5 micrograms/ml of monensin at 41 C, as observed by light and scanning electron microscopy, whereas there was no change in structure of most treated FS139 sporozoites.