Quantitative tracking of Cryptosporidium infection in cell culture with CFSE

Immunofluorescence-based assays have been developed to detect and quantitate Cryptosporidium parvum infection in cell culture. Here, we describe a method that tracks and quantifies the early phase of attachment and invasion of C. parvum sporozoites using a fluorescent dye. Newly excysted sporozoites were labeled with the amine-reactive fluorescein probe carboxyfluorescein diacetate succinimidyl esters (CFSE) using an optimized protocol. The initial invasion of cells by labeled parasites was detected with fluorescent or confocal microscopy. The infection of cells was quantified by flow cytometry. Comparative analysis of infection of cells with CFSE-labeled and unlabeled sporozoites showed that the infectivity of C. parvum was not affected by CFSE labeling. Quantitative analysis showed that C. parvum Iowa and MD isolates were considerably more invasive than Cryptosporidium hominis isolate TU502. Unlike immunofluorescent assays, CFSE labeling permitted the tracking of the initial invasion of C. parvum. Such an assay may be useful for studying the dynamics of host cell-parasite interaction and possibly for drug screening.     

Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.     

Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites

Cryptosporidium parvum is an intracellular protozoan parasite belonging to the phylum Apicomplexa, and a major cause of waterborne gastroenteritis throughout the world. Invasive zoites of apicomplexan parasites, including C. parvum, are thought to have characteristic organelles on the apical apex; however, compared with other parasites, the cytoskeletal ultrastructure of C. parvum zoites is poorly understood. Thus, in the present study, we ultrastructurally examined C. parvum sporozoites using electron microscopy to clarify the framework of invasive stages. Consequently, at the apical end of sporozoites, 3 apical rings and an electron-dense collar were seen. Two thick central microtubules were seen further inside sporozoites and extended to the posterior region. Using anti-alpha and -beta tubulin antibodies generated from sea urchin and rat brain, both antibodies cross-reacted at the apical region of sporozoites in immunofluorescent morphology. The molecular mass of C. parvum alpha tubulin antigen was 50 kDa by Western blotting and the observed apical cytoskeletal structures were shown to be composed of alpha tubulin by immunoelectron microscopy. These results suggested that C. parvum sporozoites were clearly different in their cytoskeletal structure from those of other apicomplexan parasites.     

Development of a Rapid Detection Procedure for Ctyptosporidium, Using In Vitro Cell Culture Combined with PCR

A detection, viability, and infectivity assay was developed for Cryptosporidiurn parvum. Oocysts or excysted sporozoites were inoculated onto monolayers of CaCo-2 cells grown on chamber slides. C. parvum infection was monitored by three methods: a) application of a fluorescein-labeled anti-sporozoite antibody; b) PCR of a heat-shock protein gene fragment; and c) detection of mRNA from the heat-shock protein gene by RT-PCR.     

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.     

Heterogeneous expression and functional analysis of two distinct replication protein A large subunits from Cryptosporidium parvum

Replication protein A is a single stranded DNA-binding protein that has multiple roles in eukaryotic DNA metabolism. Typically, eukaryotic replication protein A is a stable heterotrimeric complex with three subunits of 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3). We have previously cloned and characterised an RPA1 subunit from Cryptosporidium parvum, which shares high homology with other eukaryotic replication protein A 1 proteins, but lacks an N-terminal domain. Here, we have identified a second replication protein A 1 (termed CpRPA1B) from the ongoing C. parvum genome-sequencing project. The deduced protein sequence to CpRPA1B shows only 16% sequence identity with CpRPA1, indicating that two different types of RPA1 subunits are present in C. parvum. The CpRPA1B gene predicts a 75.5 kDa peptide similar in size to those of higher eukaryotes, but in contrast to the 53.9 kDa N-terminal short-type CpRPA1 protein. However, western blot analysis suggested that, although the entire CpRPA1B open reading frame might be translated, the protein may be cleaved by posttranslational modification, similar to that observed with the replication protein A 1 gene product in Plasmodium falciparum. Indirect immunofluorescence studies indicated a diffused pattern for both proteins in sporozoites. However, differential localisation was observed with CpRPA1 to the anterior region that contains the apical-complex and CpRPA1B to the central region in/or around the nuclei of the sporozoites. Both CpRPA1 and CpRPA1B full-length open reading frames were expressed for functionality assays. The CpRPA1 and CpRPA1B recombinant proteins were expressed in bacterial Escherichia coli as maltose-binding protein fusion proteins and the entire fusion proteins were assayed for their DNA-binding properties. Studies indicate that CpRPA1B binds ssDNA of >or=5 nucleotides (dT), while CpRPA1 only binds ssDNA >or=20 nucleotides (dT). This study indicates that C. parvum possesses two different types of replication protein A large subunits (replication protein A 1), both differing significantly from their hosts.     

Cryptosporidium parvum life cycle in suckling mice: a Nomarski interference-contrast study of a human-derived strain.

Cryptosporidiosis has emerged as one of the life-threatening opportunistic enteric infections in HIV-infected persons. To date, Cryptosporidium parvum is known to infect man via person-to-person or zoonotic transmission. We studied the sequential stages of the life cycle of C. parvum by Normarski interference-contrast microscopy in fresh gut specimens of newborn mice, infected with a strain derived from an AIDS patient with cryptosporidial diarrheal enteritis. Many 4- to 5-day-old suckling BALB/C mice were orally inoculated with 1 x 10(6) oocysts, obtained by acid flocculation of the patient's stools. The animals were sacrificed from 4 to 96 h post-infection and the ileum was examined microscopically. All stages of the asexual life cycle of C. parvum, from excysted sporozoites in the intestinal lumen through the development of type II mature meronts, 12- to 72-h post-infection, were documented by extemporaneous microscopic evaluation of fresh gut samples. The sexual cycle, characterized by the appearance of micro- and macrogametocytes, followed by a zygote developing into a sporulated oocyst, was documented as early 48-h post-infection. Our Nomarski interference-contrast observations on the life cycle of C. parvum yielded data comparable with those originally published by Current and Reese, and confirm the results of previous electron microscopic studies performed by several other authors.     

Sexual and Asexual Development of Cryptosporidium parvum in Five Oocyst– or Sporozoite-Infected Human Enterocytic Cell Lines

ABSTRACT. The human enterocytic cell lines Caco-2, HT29, HCT8 and the Caco-2 clones TC7 and PF11 were studied for their ability to support Cryptosporidium parvum development. Following the addition in cultures of either oocysts or excysted sporozoites, immunofluorescent and transmission electron microscopy revealed the presence of all stages of the parasite life cycle by both procedures, and no difference in the ration of infected cells was found among cell lines. More oocysts were seen in cell monolayers infected with oocysts than with sporozoites (p < 0.0001). The number of meronts observed was the same after either oocysts or sporozoites inoculation. Data suggest that the two methods yield a same cell infection rate.     

Identification of a sporozoite-specific member of the Toxoplasma SAG superfamily via genetic complementation

Toxoplasma gondii sporozoites possess an array of stage-specific antigens that are localized to the membrane and internal cellular space, as well as secreted into the primary parasitophorous vacuole. Specific labelling of viable sporozoites excysted from oocysts reveals a complex admixture of surface proteins partially shared with tachyzoites. SAG1, SRS3 and SAG3 were detected on sporozoites as well as numerous minor antigens. In contrast, tachyzoite SAG2A and B were completely absent whereas a dominant 25 kDa protein was unique to the sporozoite surface. The sporozoite gene encoding this protein was identified in tachyzoites genetically complemented with a sporozoite cDNA library and cloned via site-specific recombination into a bacterial shuttle vector. The sporozoite cDNA identified in these experiments encoded a protein with conserved structural features of the prototypical T. gondii SAG1 (P30) and shared sequence identity with surface proteins from Sarcocystis spp. This new member of the SAG superfamily was designated SporoSAG. Expression of SporoSAG in tachyzoites conferred enhanced invasion on transgenic parasites suggesting a role for this protein in oocyst/sporozoite transmission to susceptible hosts.     

The effect of microfilament inhibitor on the Cryptosporidium infection in vitro

This study was focused on the effects of microfilament inhibitor, Cytochalasin D (CD) on the invasiveness of sporozoites of Cryptosporidium spp. into the host cells. MDCK and AGS cell lines were used as host cells for C. parvum and C. muris, respectively. When MDCK cells were pretreated with CD for 1 hr before inoculation of the sporozoites, C. parvum infection was significantly inhibited when compared to the control cells. These inhibitory effects of CD on the rate of infection were dose-dependent. In addition, C. muris infection was hampered when AGS cell lines were pretreated with CD. However, the capability of invasiveness of the sporozoites into the host cells was not greatly influenced by the pretreatment of sporozoites with CD before infection. These results suggest that microfilaments of host cells, rather than parasites, play an important role for the invasion of Cryptosporidium spp.     

Cryptosporidium p30, a galactose/N-acetylgalactosamine-specific lectin, mediates infection in vitro

Cryptosporidium sp. cause human and animal diarrheal disease worldwide. The molecular mechanisms underlying Cryptosporidium attachment to, and invasion of, host cells are poorly understood. Previously, we described a surface-associated Gal/GalNAc-specific lectin activity in sporozoites of Cryptosporidium parvum. Here we describe p30, a 30-kDa Gal/GalNAc-specific lectin isolated from C. parvum and Cryptosporidium hominis sporozoites by Gal-affinity chromatography. p30 is encoded by a single copy gene containing a 906-bp open reading frame, the deduced amino acid sequence of which predicts a 302-amino acid, 31.8-kDa protein with a 22-amino acid N-terminal signal sequence. The p30 gene is expressed at 24-72 h after infection of intestinal epithelial cells. Antisera to recombinant p30 expressed in Escherichia coli react with an approximately 30-kDa protein in C. parvum and C. hominis. p30 is localized to the apical region of sporozoites and is predominantly intracellular in both sporozoites and intracellular stages of the parasite. p30 associates with gp900 and gp40, Gal/GalNAc-containing mucin-like glycoproteins that are also implicated in mediating infection. Native and recombinant p30 bind to Caco-2A cells in a dose-dependent, saturable, and Gal-inhibitable manner. Recombinant p30 inhibits C. parvum attachment to and infection of Caco-2A cells, whereas antisera to the recombinant protein also inhibit infection. Taken together, these findings suggest that p30 mediates C. parvum infection in vitro and raise the possibility that this protein may serve as a target for intervention.     

Studies on cryopreservation of Cryptosporidium parvum

Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.     

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.     

The Effects of Reducing Conditions, Medium, pH, Temperature, and Time on in Vitro Excystation of Cryptosporidium

ABSTRACT. Whereas excystation of sporozoites from oocysts of most coccidian species requires exposure to reducing conditions followed by pancreatic enzymes and bile salts, sporozoites of a bovine isolate of Cryptosporidium excysted without exposure to either reducing conditions or to pancreatic enzymes and bile salts. Without prior exposure to reducing conditions, a high percent excysted after incubation in a mixture of trypsin and bile salts in Ringer's solution; fewer excysted after incubation in tap water, even fewer after incubation in salt solutions, and none after incubation in saliva. Excystation, generally greater at pH 7.6 than at pH 6.0 and at 37°C than at 20°C, was observed as early as 1 h after incubation in water or the trypsin-bile mixture. These findings provide circumstantial evidence that oocysts of Cryptosporidium can excyst in extraintestinal sites and liberate sporozoites that can initiate autoinfection.     

Complete development of Cryptosporidium parvum in MDBK cells

Abstract Sporozoites of Cryptosporidium parvum excysted in vitro from bovine oocysts were incubated with monolayers of Madin-Darby bovine kidney cells. The extent of parasite colonisation was monitored by light microscopy and immunofluorescence. Electron microscopy confirmed the complete development and replication of C. parvum within Madin-Darby bovine kidney cells.     

The effects of reducing conditions, medium, pH, temperature, and time on in vitro excystation of Cryptosporidium

Whereas excystation of sporozoites from oocysts of most coccidian species requires exposure to reducing conditions followed by pancreatic enzymes and bile salts, sporozoites of a bovine isolate of a bovine isolate of Cryptosporidium excysted without exposure to either reducing conditions or to pancreatic enzymes and bile salts. Without prior exposure to reducing conditions, a high percent excysted after incubation in a mixture of trypsin and bile salts in Ringer's solution; fewer excysted after incubation in tap water, even fewer after incubation in salt solutions, and none after incubation in saliva. Excystation, generally greater at pH 7.6 than at pH 6.0 and at 37 degrees C than at 20 degrees C, was observed as early as 1 h after incubation in water or the trypsin-bile mixture. These findings provide circumstantial evidence that oocysts of Cryptosporidium can excyst in extraintestinal sites and liberate sporozoites that can initiate autoinfection.     

Flotillin-1 localization on sporozoites oF Eimeria tenella

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin- and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.     

Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites

Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The micronemes contained three major proteins of approximately 30, 120 and 200 kDa and the dense granules contain five major proteins in the 120-180 kDa range.