Roberta M. O'Connor

The ves Multigene Family of B. bovis Encodes Components of Rapid Antigenic Variation at the Infected Erythrocyte Surface

B. bovis, an intraerythrocytic protozoal parasite, establishes chronic infections in cattle in part through rapid variation of the polymorphic, heterodimeric VESA1 protein on the infected erythrocyte surface and sequestration of mature parasites. We describe the characterization of the ves1 alpha gene encoding the VESA1a subunit, thus providing a description of a gene whose product is involved in rapid antigenic variation in a babesial parasite. This three-exon gene, a member of a multigene family (ves), encodes a polypeptide with no cleavable signal sequence, a single predicted transmembrane segment, and a cysteine/lysine-rich domain. Variation appears to involve creation and modification or loss of a novel, transcribed copy of the gene.

Multisite Study of Cryptosporidiosis in Children with Diarrhea in India

ABSTRACT Cryptosporidium spp., a common cause of diarrhea in children, were investigated in the first multisite study in India. Diarrheal stools from hospitalized children aged <5 years from Delhi, Trichy, and Vellore were analyzed by microscopy, PCR-restriction fragment length polymorphism (RFLP), and/or sequencing at the small-subunit (SSU) rRNA and Cpgp40/15 loci for species determination and subgenotyping, respectively. Seventy of 2,579 (2.7%) children, 75% of whom were <2 years old, had cryptosporidial diarrhea as determined by microscopy. Genotyping and subgenotyping showed that Cryptosporidiumhominis was the most commonly identified species (59/67 children), and subgenotypes Ie, Ia, Ib, and Id were common in all centers. A novel C. parvum subgenotype, IIn, was identified in Vellore. Meteorological analysis revealed a higher rate of cryptosporidial positivity during hotter and drier weather in Delhi.

Characterization of a variant erythrocyte surface antigen (VESA1) expressed by Babesia bovis during antigenic variation

Babesia bovis, an intraerythrocytic, protozoal parasite of cattle, undergoes clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91-98). This ability could provide a mechanism by which the parasite escapes host immune defenses to establish chronic infection. Previous work identified two parasite-derived antigens of Mr 128,000 and 113,000 that were present on the surface of the infected erythrocyte and appeared to be associated with clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91 98). Two monoclonal antibodies (mAbs), 3F7.1H11 and 4D9.1G1, which recognize the variant erythrocyte surface antigen (VESA1) have been identified. These mAbs react only with the surface of erythrocytes infected with the B. bovis C9.1 clone in live-cell immunofluorescence assays. In both conventional and surface immunoprecipitations, the mAbs precipitate a variant antigen doublet that matches in mass the infected red blood cell (IRBC) surface antigens precipitated with bovine serum. In contrast, Western blot analysis revealed that only the Mr 128,000 polypeptide is recognized by the mAbs. Neither mAb recognizes antigenically variant progenitor or progeny parasite clones in any of the immunoassays, confirming the involvement of this antigen in rapid clonal antigenic variation. Failure to label this antigen with [9,10(n)-3H]myristic acid, [9,10(n)-3H]palmitic acid or D-[6-3H]glucosamine indicates that these polypeptides are neither N-glycosylated nor fatty acylated. Identity of the variant antigen recognized by the mAbs with that putatively identified with immune serum was confirmed by comparison of partial proteolytic digestion products. Unambiguous identification of the VESA1 antigen as a component of antigenic variation will facilitate characterization of the events leading to antigenic variation on the B. bovis-infected erythrocyte surface and its significance to parasite survival during chronic infection.

Cryptosporidiosis in patients with HIV/AIDS.

Cryptosporidium spp. are intestinal protozoan parasites of the phylum Apicomplexa, which cause diarrheal disease in humans worldwide (reviewed in [1–6]). In immunocompetent individuals, infection with this parasite may be asymptomatic or cause a self-limiting diarrheal illness. However, in immunocompromised patients such as those with HIV/AIDS Cryptosporidium spp. may cause severe, chronic and possibly fatal diarrhea and wasting. Although Cryptosporidium was discovered in 1907, it was not till 1976 that the first human cases of cryptosporidiosis were reported and not till the onset of the AIDS epidemic in the early 1980s that this parasite became widely recognized as a human pathogen (reviewed in [4,7,8]). Indeed, cryptosporidiosis was one of the original AIDSdefining illnesses and as such was associated with an increased risk of death compared to other AIDS-defining illnesses [9]. The use of highly active antiretroviral therapy (HAART) in the past 2 decades has reduced the prevalence of this disease in AIDS patients in industrialized countries [10–12]. However, the emergence of drug-resistant HIV variants and failure or discontinuation of HAART has been associated with re-emergence of Cryptosporidium spp. infection in these patients [13,14]. Even patients with advanced AIDS who are on HAART have recently been reported to have Cryptosporidium spp. infection [15]. Although antiretroviral treatment (ART) has recently become available in some developing

Expression of Cpgp40/15 in Toxoplasma gondii: a Surrogate System for the Study of Cryptosporidium Glycoprotein Antigens

ABSTRACT Cryptosporidium parvum is a waterborne enteric coccidian that causes diarrheal disease in a wide range of hosts. Development of successful therapies is hampered by the inability to culture the parasite and the lack of a transfection system for genetic manipulation. The glycoprotein products of the Cpgp40/15 gene, gp40 and gp15, are involved in C. parvum sporozoite attachment to and invasion of host cells and, as such, may be good targets for anticryptosporidial therapies. However, the function of these antigens appears to be dependent on the presence of multiple O-linked α-N-acetylgalactosamine (α-GalNAc) determinants. A eukaryotic expression system that would produce proteins bearing glycosylation patterns similar to those found on the native C. parvum glycoproteins would greatly facilitate the molecular and functional characterization of these antigens. As a unique approach to this problem, the Cpgp40/15 gene was transiently expressed in Toxoplasma gondii, and the expressed recombinant glycoproteins were characterized. Antisera to gp40 and gp15 reacted with the surface membranes of tachyzoites expressing the Cpgp40/15 construct, and this reactivity colocalized with that of antiserum to the T. gondii surface protein SAG1. Surface membrane localization was dependent on the presence of the glycophosphatidylinositol anchor attachment site present in the gp15 coding sequence. The presence of terminal O-linked α-GalNAc determinants on the T. gondii recombinant gp40 was confirmed by reactivity with Helix pomatia lectin and the monoclonal antibody 4E9, which recognizes α-GalNAc residues, and digestion with α-N-acetylgalactosaminidase. In addition to appropriate localization and glycosylation, T. gondii apparently processes the gp40/15 precursor into the gp40 and gp15 component glycopolypeptides, albeit inefficiently. These results suggest that a surrogate system using T. gondii for the study of Cryptosporidium biology may be useful.

Proteolytic Processing of the Cryptosporidium Glycoprotein gp40/15 by Human Furin and by a Parasite-Derived Furin-Like Protease Activity

ABSTRACT The apicomplexan parasite Cryptosporidium causes diarrheal disease worldwide. Proteolytic processing of proteins plays a significant role in host cell invasion by apicomplexan parasites. In previous studies, we described gp40/15, a Cryptosporidium sp. glycoprotein that is proteolytically cleaved to yield two surface glycopeptides (gp40 and gp15), which are implicated in mediating infection of host cells. In the present study, we showed that biosynthetically labeled gp40/15 is processed in Cryptosporidium parvum-infected HCT-8 cells. We identified a putative furin cleavage site RSRR↓ in the deduced amino acid sequence of gp40/15 from C. parvum and from all Cryptosporidium hominis subtypes except subtype 1e. Both human furin and a protease activity present in a C. parvum lysate cleaved recombinant C. parvum gp40/15 protein into 2 peptides, identified as gp40 and gp15 by size and by immunoreactivity with specific antibodies. C. hominis gp40/15 subtype 1e, in which the RSRR sequence is replaced by ISKR, has an alternative furin cleavage site (KSISKR↓) and was also cleaved by both furin and the C. parvum lysate. Site-directed mutagenesis of the C. parvum RSRR sequence to ASRR resulted in inhibition of cleavage by furin and the C. parvum lysate. Cleavage of recombinant gp40/15 and a synthetic furin substrate by the C. parvum lysate was inhibited by serine protease inhibitors, by the specific furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-cmk), and by calcium chelators, suggesting that the parasite expresses a Ca2+ dependent, furin-like protease activity. The furin inhibitor Dec-RVKR-cmk decreased C. parvum infection of HCT-8 cells, suggesting that a furin-like protease activity may be involved in mediating host-parasite interactions.

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of Cryptosporidium parvum to the Inner Surface of the Oocyst Wall

ABSTRACT Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidiumoocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls.

Polymorphic Mucin Antigens CpMuc4 and CpMuc5 Are Integral to Cryptosporidium parvum Infection In Vitro

ABSTRACT Cryptosporidium, a waterborne enteric parasite, is a frequent cause of diarrheal disease outbreaks worldwide. Thus far, the few antigens shown to be important for attachment to and invasion of the host cell by Cryptosporidium are all mucin-like glycoproteins. In order to investigate other antigens that could be important for Cryptosporidium host-parasite interactions, the Cryptosporidium genome databases were mined for other mucin-like genes. A single locus of seven small mucin sequences was identified on chromosome 2 (CpMuc1 to -7). Reverse transcriptase PCR analysis demonstrated that all seven CpMucs were expressed throughout intracellular development. CpMuc4 and CpMuc5 were selected for further investigation because of the significant sequence divergence between Cryptosporidium parvum and C. hominis alleles. Rabbit anti-CpMuc5 and -CpMuc4 antibodies identified several polypeptides in C. parvum lysates, suggestive of proteolytic processing of the mucins. All polypeptides were larger than the predicted molecular weight, which is suggestive of posttranslational processing, most likely O-glycosylation. In immunofluorescence assays, both anti-CpMuc4 and -CpMuc5 antibodies reacted with the apical region of sporozoites and revealed surface-exposed epitopes. The antigens were not shed during excystation but did partition into the aqueous phase of Triton X-114 extractions. Consistent with a role in attachment and invasion, CpMuc4 and CpMuc5 could be detected binding to fixed Caco-2A cells, and anti-CpMuc4 peptide antibodies inhibited Cryptosporidium infection in vitro. Sequencing of CpMuc4 and CpMuc5 from C. hominis clinical isolates identified several polymorphic alleles. The data suggest that these antigens are integral for Cryptosporidium infection in vitro and may be potential vaccine candidates.

Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro.

ABSTRACT The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. Previously, we reported that a Cryptosporidium parvum subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in C. parvum lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the C. parvum genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from C. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of ∼64 kDa and ∼48 kDa, for C. parvum lysates and proteins “shed” during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by C. parvum lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in C. parvum and for processing of gp40/15. Importantly, the recombinant prodomain inhibited C. parvum infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

Cryptosporidiosis in HIV/AIDS Patients in Kenya: Clinical Features, Epidemiology, Molecular Characterization and Antibody Responses

We investigated the epidemiological and clinical features of cryptosporidiosis, the molecular characteristics of infecting species and serum antibody responses to three Cryptosporidium-specific antigens in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients in Kenya. Cryptosporidium was the most prevalent enteric pathogen and was identified in 56 of 164 (34%) of HIV/AIDS patients, including 25 of 70 (36%) with diarrhea and 31 of 94 (33%) without diarrhea. Diarrhea in patients exclusively infected with Cryptosporidium was significantly associated with the number of children per household, contact with animals, and water treatment. Cryptosporidium hominis was the most prevalent species and the most prevalent subtype family was Ib. Patients without diarrhea had significantly higher serum IgG levels to Chgp15, Chgp40 and Cp23, and higher fecal IgA levels to Chgp15 and Chgp40 than those with diarrhea suggesting that antibody responses to these antigens may be associated with protection from diarrhea and supporting further investigation of these antigens as vaccine candidates.