Characterization of Calcium-Dependent Protein Kinases 3, a Protein Involved in Growth of Cryptosporidium parvum

Abstract

Calcium-dependent protein kinases (CDPKs) are considered promising targets for pharmaceutical intervention of cryptosporidiosis. Whole-genome sequencing has revealed the presence of several CDPKs (CpCDPKs) in Cryptosporidium parvum. In this study, we expressed recombinant CpCDPK3 encoded by the cgd5_820 gene in Escherichia coli. The biologic characteristics and functions of CpCDPK3 were examined using qRT-PCR, immunofluorescence microscopy, and in vitro neutralization assay. The expression of the cgd5_820 gene peaked in merozoites during in vitro culture while the CpCDPK3 protein was expressed in both sporozoites and merozoites. Polyclonal antibodies against CpCDPK3 showed no significant inhibitory effects on host invasion by the parasites. We assessed the inhibitory effects of 46 candidate compounds from molecular docking of CpCDPK3 on both C. parvum development and CpCDPK3 enzyme activities. One compound was identified to be effective. Results of these analyses suggest that CpCDPK3 might play an important role in the growth of C. parvum.

Characterization of Three Calcium-Dependent Protein Kinases of Cryptosporidium parvum

“…These results are consistent with the late peak expression of the CpCDPK5 gene in the lifecycle of the pathogen. They also agree with observations on CpCDPK3, which was shown to participate only in the intracellular growth of C. parvum in a previous study ( Zhang et al, 2020 ). The role of CpCDPK5 in C. parvum appears to be similar to the function of TgCDPK5 and PfCDPK5, which are orthologs of CpCDPK5.…”

“…CpCDPK1 is sensitive to these inhibitors due to the unique smaller gatekeeper in its ATP-binding pocket, while other CpCDPKs do not have this structure; thus, they are not susceptible to these compounds ( Wernimont et al, 2010 ; Artz et al, 2011 ; Zhang et al, 2014b ). In our previous study, we assessed the inhibitory effects of candidate compounds from molecular docking of CpCDPK3 on C. parvum development and CpCDPK3 enzyme activities and identified one effective compound ( Zhang et al, 2020 ). In this study, we tested 9 candidate compounds for CpCDPK4, 40 candidate compounds for CpCDPK5, and 10 candidate compounds for CpCDPK6 based on the results of molecular docking.…”

Comparative Characterization of CpCDPK1 and CpCDPK9, Two Potential Drug Targets Against Cryptosporidiosis

“…Although previous observations suggested there was no apparent correlation between anti-CpCDPK1 activities and C. parvum growth-inhibition by candidate CDPK1 inhibitors [17,35], we have provided some evidence suggesting that inhibiting the activity of CpCDPK1 can potentially affect the invasion of C. parvum. The compound F083-0116 can also inhibit the activity of CpCDPK3, which is phylogenetically related to CpCDPK1 and has similar domain structures [12]. As C. parvum development cannot be inhibited by anti-CpCDPK3 antibodies, the reduction of C. parvum growth by compound F083-0116 could be mediated through CpCDPK1 rather than CpCDPK3.…”

“…are single-copied, the multicopy name of CDPK genes illustrates the importance of CDPKs in the biology of the pathogen. Thus far, several CDPKs of C. parvum (CpCDPKs) have been characterized [6,[11][12][13]. Among them, CpCDPK1 has been most studied, with several inhibitors of the enzyme being identified [14][15][16].…”

Characterization of Calcium-Dependent Protein Kinase 2A, a Potential Drug Target Against Cryptosporidiosis

“…Similarly, Cp CDPK1, Cp CDPK4, Cp CDPK6, and Cp CDPK9 have all been shown to participate in the invasion of host cells by C. parvum ( Zhang et al, 2021 ; Su et al, 2022 ). In contrast, Cp CDPK3 and Cp CDPK5 might play some roles mostly during the growth of the parasite ( Zhang et al, 2020 ).…”