Abstract
As the invasion, egress, and growth of Cryptosporidium spp. are regulated by the calcium ion, calcium-dependent protein kinases (CDPKs) are considered potential drug targets against these pathogens. In this study, we expressed CpCDPK1 of Cryptosporidium parvum encoded by the cgd3_920 gene and CpCDPK9 encoded by the the cgd7_1260 gene in Escherichia coli, and we conducted some comparative studies with quantitative PCR, immunofluorescence staining, and in vitro neutralization assays. By immunofluorescence microscopy, CpCDPK1 was expressed over the entirety of the sporozoites, while CpCDPK9 was mainly expressed in the apical region. The expression of the cgd3_920 gene was the highest at 12 h of the in vitro culture, whereas the expression of the cgd7_1260 gene peaked between 2 h and 6 h. Polyclonal antibodies against these two CpCDPK proteins had similar neutralization efficiency on C. parvum growth, reaching approximately 40%. Of the 50 candidate compounds from the molecular docking of CpCDPK1, 10 had significant in vitro anti-cryptosporidial effects, but only one inhibited enzyme activity. For CpCDPK9, five of the forty-five candidate compounds showed significant in vitro anti-cryptosporidial effects. Results obtained from this study suggest that CpCDPK1 and CpCDPK9 might function differently in C. parvum infection.
Highlights
Cryptosporidium spp. are apicomplexan parasites responsible for cryptosporidiosis, an important diarrheal disease in humans [1]
Pure recombinant CpCDPK1 protein was obtained using Ni-NTA purification through the His-tag incorporated (Figure 1D, top panel), while pure CpCDPK9 protein was obtained by gel extraction (Figure 1D, bottom panel)
Upper panel for CpCDPK1: lane M, molecular weight markers; lane 1, lysate from bacterial culture transformed with the recombinant plasmid with no isopropyl b-D-1-thiogalactopyranoside (IPTG) induction; lane 2, lysate from similar bacteria culture induced with IPTG for 2 h; lane 3, lysate from bacteria culture induced with IPTG for 8 h, with the expected product being indicated by an arrow
Summary
Cryptosporidium spp. are apicomplexan parasites responsible for cryptosporidiosis, an important diarrheal disease in humans [1]. The diarrhea lasts for 1–2 weeks [2]. Cryptosporidiosis can lead to prolonged, life-threatening diarrhea in immunocompromised persons, such as AIDS patients. Among the over 40 established Cryptosporidium species, C. parvum and C. hominis are the dominant. Cryptosporidium species responsible for human cryptosporidiosis [3]. As C. parvum can infect farm animals, it is a zoonotic pathogen. There are few effective drugs against cryptosporidiosis. The only one approved by the U.S Food and Drug Administration is nitazoxanide. Nitazoxanide, is not effective in treating cryptosporidiosis in immunocompromised patients or malnourished children, probably because it cannot block the infection [4]. The development of anti-parasitic drugs is urgently needed for cryptosporidiosis
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