Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvum attachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15 that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15 revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy of Cpgp40/15 in the C. parvum genome, and this gene does not contain introns. Our data indicate that the two Cpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis.Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal disease worldwide (7,36). Cryptosporidial infection is asymptomatic or self-limiting in immunocompetent hosts, but it may be chronic and lifethreatening in immunocompromised patients such as those with AIDS. In children in developing countries, C. parvum infection has been reported to be associated with persistent diarrheal disease, which may result in subsequent growth impairment (8). Recently, this parasite has been implicated as the causative agent of numerous outbreaks of waterborne diarrheal disease (6). There is currently no specific therapy approved for the treatment of cryptosporidiosis.Infection with C. parvum is initiated by ingestion of oocysts, which undergo excystation to release sporozoites. Sporozoites attach to and invade intestinal epithelial cells, where the parasite undergoes further intracellular development through asexual as well as sexual cycles. Merozoites released into the lumen during the asexual cycle can attach to and invade adjacent epithelial cells and thus maintain intracellular replication. The molecular mechanisms involved in the pathogenesis of cryptosporidiosis and the specific molecules that mediate C. parvum-host cell interactions are unknown. A number of C. parvum proteins have been implicated in attachment and in...

Cryptosporidium sp. is a significant cause of diarrheal disease, particularly in human immunodeficiency virus (HIV)-infected patients in developing countries. We recently cloned and sequenced several alleles of the highly polymorphic single-copy Cryptosporidium parvum gene Cpgp40/15. This gene encodes a precursor protein that is proteolytically cleaved to yield mature cell surface glycoproteins gp40 and gp15, which are implicated in zoite attachment to and invasion of enterocytes. The most-striking feature of the Cpgp40/15 alleles and proteins is their unprecedented degree of sequence polymorphism, which is far greater than that observed for any other gene or protein studied in C. parvum to date. In this study we analyzed nucleic acid and amino acid sequence polymorphism at the Cpgp40/15 locus of 20 C. parvum isolates from HIV-infected South African children. Fifteen isolates exhibited one of four previously identified genotype I alleles at the Cpgp40/15 locus (Ia, Ib, Ic, and Id), while five displayed a novel set of polymorphisms that defined a new Cpgp40/15 genotype I allele, designated genotype Ie. Surprisingly, only 15 of these isolates exhibited concordant type I alleles at the thrombospondin-related adhesive protein of Cryptosporidium and Cryptosporidium oocyst wall protein loci, while five isolates (all of which displayed Cpgp40/15 genotype Ic alleles) displayed genotype II alleles at these loci. Furthermore, the last five isolates also manifested chimeric genotype Ic/Ib or Ic/II alleles at the Cpgp40/15 locus, raising the possibility of sexual recombination within and between prototypal parasite genotypes. Lastly, children infected with isolates having genotype Ic alleles were significantly older than those infected with isolates displaying other genotype I alleles.

The protozoan parasite Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells by C. parvum sporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putative C. parvum adhesion-and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralized C. parvum infection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains ␣-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and ␣-N-acetylgalactosaminespecific lectins strongly implicate these proteins and their glycotopes as playing a role in C. parvum-host cell interactions.Cryptosporidium parvum, an intestinal Apicomplexan parasite, is a significant cause of diarrheal disease worldwide (15,17). In immunocompetent individuals, the disease is usually self-limiting, but it may be chronic and life threatening in immunocompromised patients such as those with AIDS. Recently, the parasite has gained notoriety as the causative agent of numerous outbreaks of waterborne diarrheal disease. There is currently no effective specific therapy approved for disease caused by this parasite.Infection is initiated by ingestion of oocysts, which undergo excystation to release sporozoites. Attachment of sporozoites to epithelial cells and subsequent invasion of the host cell membrane are crucial primary steps in the pathogenesis of cryptosporidiosis. The ultrastructural aspects of attachment and invasion have been characterized in detail (10,24,33,34). Sporozoites attach to host cells by their anterior pole. Attachment is followed by invagination of the host cell plasma membrane, which extends along the surface of the sporozoite and eventually completely surrounds it, leading to formation of a parasitophorus vacuole where the parasite undergoes further development in a unique intracellular but extracytoplasmic location.Using in vitro models of sporozoite attachment to epithelial cells, we previously showed that attachment was dose and time dependent and was influenced by pH, divalent cations, and the degree of differentiation of host cells (20,22). Further, attachment could be inhibited by polyclo...

Chagas disease is a chronic infection that kills approximately 12,000 people a year. Mass migration of chronically infected and asymptomatic persons has caused globalization of Chagas disease and has made nonvectorial infection, including vertical and blood-borne transmission, more of a threat to human communities than vectorial infection. To control transmission, it is essential to test all pregnant women living in endemic countries and all pregnant women having migrated from, or having lived in, endemic countries. All children born to seropositive mothers should be tested not only within the first month of life but also at ~6 months and ~12 months of age. The diagnosis is made by identification of the parasite in blood before the age of 6 months and by identification of the parasite in blood and/or positive serology after 10 months of age. Follow up for a year is essential as a significant proportion of cases are initially negative and are only detected at a later stage. If the condition is diagnosed and treated early, the clinical response is excellent and the majority of cases are cured.

The study of genomic organization and regulatory elements of rRNA genes in metazoan paradigmatic organisms has led to the most accepted model of rRNA gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial eukaryotes have also been studied in considerable detail and their atypical structures have been considered as exceptions. However, it is likely that these organisms have preserved variations in the organization of a versatile gene that may be seen as living records of evolution. Here, we review the organization of the main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These genes are reiterated in the genome of microbial eukaryotes and may be coded alone, in tandem repeats, linked to each other or linked to other genes. They may be found in the chromosome or extrachromosomally in linear or circular units. rDNA coding regions may contain introns, sequence insertions, protein-coding genes or additional spacers. The 5S rDNA can be found in tandem repeats or genetically linked to genes transcribed by RNA polymerases I, II or III. Available information from about a hundred microbial eukaryotes was used to review the unexpected diversity in the genomic organization of rRNA genes.

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Cryptosporidium spp. are ubiquitous waterborne parasites responsible for outbreaks of diarrheal disease worldwide [1]. The infection is self-limiting in immunocompetent individuals, but in immunocompromised individuals the infection can develop into a chronic, debilitating, and sometimes life-threatening disease [2]. In malnourished children, cryptosporidiosis is associated with growth and developmental delays [1]. Currently, there are no vaccines to prevent cryptosporidiosis, and nitazoxanide, the only approved drug in the US [3] is not effective in AIDS patients [4]. Because the parasite cannot be propagated in vitro or genetically manipulated, data that would permit targeted drug design or vaccine development are sorely lacking.Ingested Cryptosporidium oocysts excyst in the intestine, releasing sporozoites that attach to and invade intestinal epithelial cells, initiating the lifecycle [5]. The parasite undergoes two rounds of asexual reproduction, in each cycle producing merozoites that invade new epithelial cells, before entering the sexual cycle and producing oocysts. One research focus has been to identify the antigens on the invasive zoite stages that allow the parasite to attach to and invade the epithelial cell with the goal of preventing these parasite-host cell interactions. The best characterized of the zoite antigens is Cpgp40/15, (also called Cp17, gp15/45/60 or S60) [6][7][8][9][10], a mucin like glycoprotein antigen that is synthesized as a single precursor protein and proteolytically cleaved into the mature glycoproteins, gp40 and gp15 [11]. gp15 is anchored in the sporozoite membrane by a glycosylphosphatidyl inositol (GPI) moiety [12], while the gp40 glycoprotein does not contain any predicted transmembrane domains or GPI anchors and is predicted to be soluble [7]. However, gp40 has been shown to bind intestinal epithelial cells in a dose dependent and saturable manner [7] suggesting that this antigen recognizes a host cell receptor. If this interaction is important for zoite attachment and invasion, then gp40 must have some mechanism of associating with the parasite membrane. In these studies we explore the possibility that gp40 and gp15 associate to form a protein complex capable of linking zoite and host cell surfaces.* Corresponding Authors NEMC Box 041, 750 Washington St, Boston, MA 02111, e-mail: roconnor@tufts-nemc.org; hward@tufts-nemc.org, tel ROC: 617 636 2684, HW: 617 636 7022, fax: 617 636 5292. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To generate gp40 specific antiserum, recombinant (r)gp40 fusion protein [7] was purified via the His...