Identification and characterization of a novel calcium-activated apyrase from Cryptosporidium parasites and its potential role in pathogenesis

Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.     

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.     

Ultrastructural study of asexual development of Cryptosporidium parvum in a human intestinal cell line.

The lack of a well-defined in vitro model of Cryptosporidium infection has severely hampered research on the biology of parasitic invasion of the host cell and on intracellular development of the parasite. In vitro infection of the differentiated human enterocyte cell line HT29.74 was studied by electron microscopy to detect changes in parasite and host cell morphology. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned differentiated HT29.74 cells. Parasites and infected cells were evaluated by transmission electron microscopy at 20 min, 1 h, 6 h, 24 h and 7 days. Sporozoite invagination within the epithelial cell microvilli and subsequent penetration was evident at 1 h. At 6 h, the development of a dense band and feeder layer was visible. Development of the trophozoite into a schizont occurred over 24 h. Micronemes and dense granules were clearly visible within sporozoites and merozoites. Organization of vacuoles within the cytoplasm of the host cell was evident below the dense band. A sexual Cryptosporidium development in vitro was morphologically no different from initial development in vivo. In vitro infection of HT29.74 cells provides an excellent model to study parasite-host cell interaction and asexual parasite development.     

Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits.

Cell walls of Clostridium thermohydrosulfuricum and C. thermosaccharolyticum have a two-layered structure, consisting of a thin, lysozyme-sensitive murein layer and a surface (S) layer composed of hexagonally or tetragonally arranged subunits. The subunits can be removed from the murein layer by treatment of cell wall preparations, are composed of a fragile, pH-sensitive monolayer of macromolecular subunits. In both organisms the first stage of the cell division process involves only the plasma membrane and the murein layer. During the subsequent cell separation, a surplus of S-layer subunits appears at the site of division, and consequently the newly formed cell poles remain completely covered by the s layer throughout the separation process. In autolyzed cells an additional layer of subunits assembles on extended areas of the inside of the mucopeptide layer. These observations indicate that the biological function of the S layer depends on its ability to maintain a complete covering of the cell surface at all stages of cell growth and division.     

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 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.     

Highlights and summaries of the 11th International Workshops on Opportunistic Protists

The 11th in the series of International Workshops on Opportunistic Protists (IWOP-11) was held in August 2010 on the Big Island of Hawaii. These meetings are devoted to agents of infections that cause serious problems in AIDS patients and other individuals with defective immune systems. International Workshops on Opportunistic Protists serves as a forum for exchange of current research information on Pneumocystis, Cryptosporidium and the Microsporidia, Toxoplasma, free-living amoebae, kinetoplastid flagellates and other pathogens that are particularly pathogenic in immunodeficient hosts. Studies on interactions between host and pathogen, especially host responses, were highlighted in this year's symposium. The lack of in vitro cultivation methods for luxuriant growth of Pneumocystis, Cryptosporidium and the Enterocytozoon bieneusi remains a major hindrance to understanding the basic biology of these organisms and precludes genetic manipulations. However, slow but steady progress is being achieved by hard work including data mining of some completed or partially completed genome sequencing of several IWOP organisms. Of great concern is evidence for dramatic decline in research funding for these pathogens and the lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms that can provide critical insight into emerging and reemerging pathogens.     

Prevalence and identity of Cryptosporidium spp. in pig slurry

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.     

Geosiphon pyriforme,an Endosymbiotic Association of Fungus and Cyanobacteria: the Spore Structure Resembles that of Arbuscular Mycorrhizal (AM) Fungi

The zygomycete Geosiphon pyriforme is the only known endocyanosis of a fungus. The Nostoc spp. filaments are included in photosynthetically active and nitrogen fixing, multinucleated bladders, which grow on the soil surface. The spores of the fungus are white or slightly brownish. They are about 250 μm in diameter and develop singly on hyphal ends or, less frequently, intercalarly. The wall of the spores consists of a thin innermost layer, a laminated inner layer with a thickness of about 10–13 μm, and an evanescent outer layer. The laminated layer is composed of helicoidally arranged microfibrils, and is separated from the evanescent outer layer by a thin electron-dense sublayer. Polarisation microscopy indicates the occurrence of chitin. Shape and wall ultrastructure of the Geosiphon spores and their cytoplasm resemble that of Glomus spores, but are different from that of other genera of the Glomales and Endogonales. Germination occurs by a single thick hyphal outgrowth directly through the spore wall. Like various AM forming fungi, Geosiphon pyriforme contains endocytic bacteria-like organisms, which are not surrounded by a host membrane. Our observations indicate that Geosiphon is a potential AM fungus.     

Mechanism of C. trachomatis attachment to eukaryotic host cells.

A novel trimolecular mechanism of microbial attachment to mammalian host cells was characterized for the obligate intracellular pathogen Chlamydia trachomatis. Using purified glycosaminoglycans (GAGs) and specific GAG lyases, we demonstrated that a heparan sulfate-like GAG present on the surface of chlamydia organisms is required for attachment to host cells. These observations were supported by inhibition of attachment following binding of heparan sulfate receptor analogs to chlamydiae and by demonstrating that chlamydiae synthesize a unique heparan sulfate-like GAG. Furthermore, exogenous heparan sulfate, as an adhesin analog, restored attachment and infectivity to organisms that had lost these attributes following treatment with heparan sulfate lyase. These data suggest that a GAG adhesin ligand mediates attachment by bridging mutual GAG receptors on the host cell surface and on the chlamydial outer membrane surface.     

Cellular biology of Cryptosporidium parvum

With the emergence of Cryptosporidium parvum as a major pathogen encountered in human and veterinary clinical practice, a need for increased knowledge of the cellular- and immuno-biology of this Apicomplexan parasite has developed. Initial work has used paradigms taken from other Apicomplexans, especially Plasmodium, Toxoplasma and Eimeria, as a starting point. In this article, Carolyn Petersen discusses the observation that in these organisms, molecular targets of antibodies (which have protective value, in vivo, against disease) have frequently been located in the apical complex or on the surface of the invasive stages of the parasite and appear to mediate biologically crucial processes including motility, attachment to the host cell, modification of the host membrane, and entry into the host cell. Molecular-biology approaches to the study of enzymes and of structural proteins which mediate motility are also considered. Invasion mechanisms, biochemical pathways and motility may involve molecules that will prove susceptible to immunotherapeutic or chemotherapeutic interruption of cryptosporidiosis.     

Chlamydia--host cell interactions: recent advances on bacterial entry and intracellular development

Bacteria of the Chlamydiales order are very successful intracellular organisms that grow in human and animal cells, and even in amoebae. They fulfill several essential functions to enter their host cells, establish an intracellular environment favorable for their multiplication and exit the host cell. They multiply in a unique organelle called the inclusion, which is isolated from the endocytic but not the exocytic pathway. A combination of host cell factors and of proteins secreted by the bacteria, from within the inclusion, contribute to the establishment and development of this inclusion. Here we review recent data on the entry mechanisms and maturation of the inclusion.     

Molecular analysis of the 18S rRNA gene of Cryptosporidium serpentis in a wild-caught corn snake (Elaphe guttata guttata) and a five-species restriction fragment length polymorphism- based assay that can additionally discern C. parvum from C. wrairi

An adult wild-caught corn snake (Elaphe guttata guttata) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-microm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for Cryptosporidium spp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as Cryptosporidium serpentis. Until recently, studies on snake Cryptosporidium have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of C. serpentis (GenBank accession no. AF093499, AF093500, and AF093501 [L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the C. serpentis (Snake) sequence (AF093499) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of Cryptosporidium at this locus was developed. This assay, which uses SpeI and SspI, complements a previously reported assay by additionally distinguishing the bovine strain of Cryptosporidium from Cryptosporidium wrairi.     

The other mediator for adherence of Haemophilus influenzae organisms without involvement of fimbriae.

The number of the nontypeable Haemophilus influenzae (HiN) organisms that adhered to the primary mouse fetal lung cells was significantly more than type b Haemophilus influenzae (Hib) organisms. The average number of HiN organisms adherent to host cells was 2,291/100 host cells (range, 1,654-3,182), but that of Hib was markedly reduced to 147/100 host cells (range, 102-238). In this case, P value was less than 0.05 by using a paired Student t-test. The sonicated extract from HiN TMS11 organisms inhibited adherence of H. influenzae TMS11 organisms to monolayer at 76.3% and it inhibited adherence of Hib TMS24 organisms at 92.3%. This result indicates that a mediator existing on the surface of HiN organisms may be the same as that on type b organisms. The number of detected organisms in broncho and lung tissues 3 days after intranasal infection with HiN strains was significantly greater than that in infection with Hib strains. Therefore, in vitro adhesive capacity of H. influenzae organisms was correlative to infectivity by intranasal injection.     

Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode

The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritin-conjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretion along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior.     

Revisiting the extracellular lifestyle

In microbiology textbooks infectious agents are traditionally classified as intracellular or extracellular pathogens depending on whether they multiply inside or outside host cells. In recent literature an increasing number of extracellular pathogens are described in close apposition with the host cell surface embedded in plasma membrane folds, making it difficult to classify them as strictly extracellular pathogens. This review further explores this emerging new lifestyle category tentatively named 'epicellular' in reference to earlier work describing the location of the parasite Cryptosporidium parvum. The lifestyles of three diverse such pathogens were examined: the parasite Cryptosporidium parvum, the Gram-negative bacterium Neisseria meningitidis and the human T cell leukaemia virus type 1 (HTLV-1). The specific cellular location, the mechanisms of adhesion, the induction of plasma membrane folds and the subsequent functional consequences will be compared. Although current knowledge suggests different underlying mechanisms, a concept that emerges is that the particular location of these pathogens has important functional consequences for the pathogens in terms of nutrient acquisition, immune escape, resistance to mechanical stress and transmission. Re-examining the lifestyle of other classically extracellular pathogens might thus shed a new light on the way pathogens interact with cells and point to new areas of investigation.     

Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater.

Giardiasis and cryptosporidiosis are diseases caused by the protozoan parasites Giardia lamblia and Cryptosporidium parvum. Waterborne transmission of these organisms has become more prevalent in recent years, and regulatory agencies are urging that source and finished water be screened for these organisms. A major problem associated with testing for these organisms is the lack of reliable methodologies and baseline information on the prevalence of these parasites in various water sources. Our study addressed both of these issues. We evaluated the presence and reduction of Giardia cysts and Cryptosporidium oocysts in sewage effluent by a combination of indirect fluorescent antibody (IFA) staining and PCR. Our results indicated a 3-log reduction of Giardia cysts and a 2-log reduction of Cryptosporidium oocysts through the sewage treatment process as determined by IFA. We developed a nested PCR to detect Cryptosporidium oocysts and used a double PCR to detect Giardia cysts. A 100% correlation was noted between IFA and PCR detection of Giardia cysts while correlation for Cryptosporidium oocysts was slightly less. On the basis of these results, PCR may be a useful tool in the environmental analysis of water samples for Giardia and Cryptosporidium organisms.     

The morphology of the lung of a tropical terrestrial slug Trichotoxon copleyi (Mollusca: Gastropoda: Pulmonata): A scanning and transmission electron microscopic study

The lung of the slug Trichotoxon copleyi is located in the body mantle where it opens to the outside through an adjustable pneumostome situated on the right side. The respiratory surface of the lung is profusely vascularized by anastomosing 'blood' capillaries. A thick muscular layer surrounds the gas exchange surface and may be involved in the efficient rhythmic ventilatory contraction of the lung. Externally an epithelial layer made up of large secretory cells with numerous microvilli overlies the muscular layer. The surface of the lung is made up of squamous cells with an abundance of microvilli and clustered goblet cells interspersed occasionally. The squamous cells cover the notably thin air–blood barrier which is extremely attenuated, particularly in some areas. The goblet cells contain a centrally located nucleus and numerous intracytoplasmic granules, presumably precursors of the mucus that covers the surface of the mantle cavity.
All the essential morphological prerequisites for efficient gas exchange such as an extensive surface and a thin blood–gas barrier were observed in the lung of Trichotoxon. It was concluded that this molluscan lung is structurally and functionally a true lung in all respects.     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.     

Electron microscopy studies of the limiting layers of the rickettsia Coxiella burneti.

Surface layers of Coxiella burneti studied at a high resoulution reveal a plasma membrane and an outer surface membrane 6 to 7 nm thick, and a thin, moderately electron-dense intermediate layer associated with the inner surface of the outer membrane of many cells. This layer appears to be unaffected by lysozyme treatment. Ruthenium red staining was used to delineate a layer of filamentous material external to the outer membrane; this fuzzy layer has a mean thickness of 20 nm and is not often seen on the surface of cells prepared by conventional means. Both antigenic phase I and II cells show a ruthenium red-binding surface layer. It is suggested that this fuzzy layer may be, among other possibilities, a highly branched mucopolysaccharide.