María-Teresa Bonafonte

CYTOKINE EXPRESSION AND SPECIFIC LYMPHOCYTE PROLIFERATION IN TWO STRAINS OF CRYPTOSPORIDIUM PARVUM-INFECTED γ-INTERFERON KNOCKOUT MICE

Differences in the immune response between 2 strains of interferon-γ knockout mice (BALB/c-GKO and C57BL/6-GKO) infected with Cryptosporidium parvum were examined because the course of infection among these 2 strains is markedly different. Infection of the BALB/c-GKO with C. parvum (2 × 106 oocysts/mouse) resulted in slight weight loss, oocyst shedding, and recovery from infection by 2 wk postinfection (PI). Infection with 100 oocysts in the C57BL/6-GKO mice resulted in significant weight loss, oocyst shedding, and death by day 10 PI. Splenocytes from infected mice were able to proliferate in a dose-dependent manner to soluble C. parvum-sporozoite antigen (SAg). In vitro stimulation with SAg resulted in an increase in interleukin (IL)-2, IL-4, IL-5, and tumor necrosis factor-α mRNA cytokine expression from splenocytes of infected BALB/c-GKO mice. In contrast, only IL-5 mRNA expression was increased in the splenocytes from C. parvum-infected C57BL/6-GKO mice. Phenotypic analysis indicated no significant differences in the splenic cell populations. Previous studies indicated that susceptibility to C. parvum is dependent on CD4+ T cells and interferon-γ production. The present study indicates that although both of these strains of knockout mice become infected with C. parvum, resolution of infection may be in part dependent on the expression of Th2 cytokines.

Isolation of the gene coding for elongation factor-1α in Cryptosporidium parvum

A gene encoding for Cryptosporidium parvum (C. parvum) elongation factor 1alpha (EF-1alpha) was isolated and sequenced from a cDNA expression library. The recombinant protein cross-reacted with a monoclonal antibody that was raised to a sporozoite cell surface antigen. The gene encoded a 435 amino acid protein with a predicted molecular weight of 48.1 kDa. The predicted C. parvum EF-1alpha protein sequence showed extensive homology with the EF-1alpha proteins of other eukaryotic organisms and included three conserved sequence motifs implicated in GTP binding.

Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis.

Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene. We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E. intestinalis, including a P-gp.

Characterization of an α-Tubulin Gene of Cryptosporidium parvum

A gene encoding an α‐tubulin of Cryptosporidium parvum was isolated and characterized. It had no introns, and encoded a 441‐amino acid protein whose predicted ORF represented a typical α‐tubulin protein with a MW of 50.5 kDa. This tubulin had an amino acid sequence similarity with Apicomplexa Plasmodium falciparum and Toxoplasma gondii higher than 88% and shared a number of conserved motifs.

Cloning and Expression of Sporozoite and Oocyst Cryptosporidium parvum Recombinant Proteins.

MARIA-TERESA BONAFONTE,’p2 ROBERT M. LLOYD 5%’’ K. DEANNA GARMON,” XIANGDONG YOU,’J MICHAEL J. ARROWOOD ’ RAYMOND F. SCHINAZI ,’,’ AhD JAN R. MEAD.’” ‘Emory University, A t l m k ~ , GA 30033, zVA hledical Center, Decatur, GA 30033, and ’Centers for Disease Control and Prevention, AthnkZ, GA 30341. Cryptosporidiurn parvum (C. parvurn) is a protozoan parasite that infects the intestinal epithelium of mammals. Sera from infected humans and animals recognize the zoite and oocyst antigens and genes encoding several of these proteins have been expressed i n Escherichia coli (E. coli). The identification and characterization of new parasite proteins is necessary to study the immune response against C. parvum antigens. Our goal was to clone and to express recombinant proteins for use in immunologic assays. MATERIALS AND METHODS. A cDNA library was screened with murine monoclonal antibodies against parasite sporozoites and oocysts and several recombinant clones were isolated. Immunoreactive clones (C6C1 a sporozoite surface antigen) and (3-1 an oocyst wall antigen) were sequenced in both directions using an automated 377 ABI Sequencer. C6C1 and 3-1 were PCR amplified and yielded a 206 and 2019 base pair fragments, respectively. C6C1 and 3-1 were cloned in-frame into the BamHI site of pCEX-2T (Pharmacia) and BamHI-Nor1 sites of @EX-4T-2 (Pharmacia) respectively. Recombinant plasmids were used to transform E. coli competent cells (DH5a or JM109). Further screening of the cDNA library yielded the isolation and identification of two new immunoreactive clones (1Al and 2A1). 1Al and 2A1 resulted in a 2000 base pair insert, their entire sequence is being determined. An overnight culture of E. coli transformed with @EX (control without insert) and recombinant plasmid was diluted 150 in fresh Luria Broth medium supplemented with 100 pg/ml ampicillin. ’The Tacpromoter was induced by adding isopropyl-8-D-thiogalactoside to a final concentration of 0.1 mM. Lysis of the cells was performed using the sarcosyl method [l]. Approximately 5-10 pl from each extract were analyzed by SDS-PAGE gel [3]. Proteins were visualized by staining the gel with Coomassie blue. Protein extracts were electroblotted onto nitrocellulose membranes and immunoreactivity was analyzed by Western blot [4] using absorbed monoclonal mouse or polyclonal rabbit antibodies (Fig. 1).