Single-chain variable fragment antibodies selected by phage display against the sporozoite surface antigen P23 Of Cryptosporidium parvum

Cryptosporidium parvum, an apicomplexan parasite transmitted via animal fecal wastes, is the causative agent of cryptosporidiosis. Clones were selected from 2 synthetic na?ve human single-chain variable fragment (scFv) phagemid libraries that bound to the recombinant P23 protein of C. parvum. Panning the Tomlinson I and J phagemid libraries resulted in 6 distinct clones. Two clones had full-length scFv sequences, while the remaining clones were either truncated or missing a section of the heavy chain. Despite these differences, all clones were able to detect both native C. parvum proteins and recombinant P23. None of the selected clones cross-reacted with Escherichia coli, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus, Giardia lamblia (cysts or trophozoites), or with S16, another dominant surface antigen on C. parvum sporozoites. Clones expressed as the scFv-gIIIp fusion construct in soluble form detected C. parvum. Panning from na?ve libraries is a useful method for isolation and identification of recombinant antibodies that have the potential for use in pathogen detection and immunotherapy.     

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.     

Cryptosporidium parvum attachment to and internalization by human biliary epithelia in vitro: a morphologic study

To explore the mechanisms by which Cryptosporidium parvum infects epithelial cells, we performed a detailed morphological study by serial electron microscopy to assess attachment to and internalization of biliary epithelial cells by C. parvum in an in vitro model of human biliary cryptosporidiosis. When C. parvum sporozoites initially attach to the host cell membrane, the rhoptry of the sporozoite extends to the attachment site; both micronemes and dense granules are recruited to the apical complex region of the attached parasite. During internalization, numerous vacuoles covered by the parasite's plasma membrane are formed and cluster together to establish a preparasitophorous vacuole. This preparasitophorous vacuole comes in contact with host cell membrane to form a host cell-parasite membrane interface, beneath which an electron-dense band begins to appear within the host cell cytoplasm. Simultaneously, host cells display membrane protrusion along the edge of the host cell-parasite membrane interface, resulting in the formation of a mature parasitophorous vacuole that completely covers the parasite. During internalization, vacuole-like structures appear in the apical complex region of the attached sporozoite, which bud out into host cells. A tunnel directly connecting the parasite to the host cell cytoplasm forms during internalization and remains when the parasite is totally internalized. Immunoelectron microscopy showed that sporozoite-associated proteins were localized along the dense band and at the parasitophorous vacuole membrane. These morphological observations provide evidence that secretion of parasite apical organelles and protrusion of host cell membrane play an important role in the attachment and internalization of host epithelial cells by C. parvum.     

Cryptosporidium parvum: identification of a new surface adhesion protein on sporozoite and oocyst by screening of a phage-display cDNA library

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.     

Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.     

甜菜黑色焦枯病毒外壳蛋白与病毒致病性的关系

利用RT-PCR方法,构建获得了由T7RNA聚合酶启动子驱动的甜菜黑色焦枯病毒(BBSV)全长cDNA克隆pUBF52.摩擦接种苋色藜(Chenopodiumamaranticolor)后,体外转录产物可导致与野生病毒相同的枯斑症状,蛋白质印迹和RNA印迹检测也都证明了转录产物的侵染活性.构建了BBSVp24基因的原核表达载体pECP1,转化大肠杆菌BL21后的诱导表达产物能够与BBSV的抗血清呈现特异性反应,表明该基因编码产生BBSV的外壳蛋白(CP).以pUBF52为模板,分别构建了BBSVCP基因的移码突变体和不同程度的缺失突变体.侵染性检测表明,CP基因的移码突变对BBSV在苋色藜上所导致的枯斑症状及病毒RNA在寄主体内的积累基本没有影响,但CP基因的大部或完全缺失会使体内病毒RNA的积累水平大大降低,其中CP基因完全缺失的突变体转录物接种苋色藜后仅能够产生很轻的枯斑症状.将绿色荧光蛋白(GFP)基因和葡糖苷酸酶(GUS)基因分别与BBSVCP基因的5′端融合,构建了表达载体pBGFP和pBGUS.摩擦接种苋色藜叶片后可观察到GFP或GUS基因的表达,为探索利用BBSV作为外源蛋白的表达载体奠定了基础.