Abstract
Cryptosporidium parvum is a major cause of moderate-to-severe diarrhea in humans and animals. Its compact genome contains 22 genes encoding divergent insulinase-like proteases (INS), which are poorly characterized. In this study, two small members of this family, INS-21 encoded by cgd7_2080 and INS-23 encoded by cgd5_3400, were cloned, expressed, and characterized to understand their functions. Recombinant INS-21 and INS-23 were expressed in Escherichia coli and polyclonal antibodies against these two proteins were prepared. The cgd7_2080 gene had a high transcription level during 0–2 h of in vitro C. parvum culture, while cgd5_3400 was highly transcribed at 0–6 h. INS-21 was mostly located in the apical region of sporozoites and merozoites whereas INS-23 was found as spots in sporozoites and merozoites. The immunoelectron microscopy confirmed the expression of INS-21 in the apical region of sporozoites while INS-23 appeared to be expressed in the dense granules of sporozoites. The neutralization efficiency was approximately 35%, when the cultures were treated with anti-INS23 antibodies. These results suggest that INS-21 and INS-23 are expressed in different organelles and might have different functions in the development of C. parvum.
Highlights
Cryptosporidium spp. are common protozoan pathogens, causing the enteric disease cryptosporidiosis in humans and animals at early age (Kotloff, 2017)
Data from the present study suggest that insulinase-like proteases (INS)-21 and INS23 may have slightly different roles in the invasion and development of C. parvum
Despite some crossreactivity of anti-INS-21 antibodies to recombinant INS-23, immunofluorescence and immunoelectron microscopy studies with these antibodies suggest that INS-21 is expressed in micronemes of sporozoites and possibly merozoites, while INS-23 is likely expressed in dense granules of these invasive stage
Summary
Cryptosporidium spp. are common protozoan pathogens, causing the enteric disease cryptosporidiosis in humans and animals at early age (Kotloff, 2017). Approximately 8.6% of global child mortality is caused by diarrheal diseases (Liu L. et al, 2016), with Cryptosporidium as one of the top five pathogens for moderate-to-severe diarrhea in children under 5 years in developing countries (Khalil et al, 2018; Platts-Mills et al, 2018; Kotloff et al, 2019). Nitazoxanide is the only drug approved by FDA for the treatment of cryptosporidiosis. It has only limited efficacy in infants and immunocompromised individuals who have the most urgent need of treatment (Bhalchandra et al, 2018).
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