Characterization of Three Calcium-Dependent Protein Kinases of Cryptosporidium parvum

In Cryptosporidium spp., calcium-dependent protein kinases (CDPKs) are considered promising targets for the development of pharmaceutical interventions. Whole-genome sequencing has revealed the presence of 11 CDPKs in Cryptosporidium parvum (CpCDPKs). In this study, we expressed recombinant CpCDPK4, CpCDPK5, and CpCDPK6 in Escherichia coli. The biological characteristics and functions of these CpCDPKs were examined by using quantitative reverse transcription PCR (qRT-PCR), immunofluorescence microscopy, and an in vitro neutralization assay. The expression of the CpCDPK4 gene peaked at 12 h post-infection, the CpCDPK5 gene peaked at 12 and 48 h, and the CpCDPK6 gene peaked at 2–6 h. CpCDPK4 protein was located in the anterior and mid-anterior regions of sporozoites, and CpCDPK5 protein was located over the entire sporozoites, while CpCDPK6 protein was expressed in a spotty pattern. Immune sera of CpCDPK4 and CpCDPK6 exhibited significant inhibitory effects on host cell invasion, while the immune sera of CpCDPK5 had no effects. These differences in protein localization, gene expressions, and neutralizing capacities indicated that the CpCDPK proteins may have different roles during the lifecycle of Cryptosporidium spp.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2020.00907 ◽

2020 ◽

Vol 11 ◽

Author(s):

Qiang Zhang ◽

Yaqiong Guo ◽

Na Li ◽

Yu Li ◽

Jiayuan Su ◽

...

Keyword(s):

Protein Kinases ◽

Cryptosporidium Parvum ◽

Calcium Dependent ◽

Dependent Protein ◽

Calcium Dependent Protein Kinases

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Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

Infection and Immunity ◽

10.1128/iai.01173-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1262-1273 ◽

Cited By ~ 35

Author(s):

Shaojun Long ◽

Qiuling Wang ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Protein Kinases ◽

Gene Deletion ◽

Cyst Formation ◽

Content Type ◽

Calcium Dependent ◽

Targeted Gene Deletion ◽

Dependent Protein ◽

Calcium Dependent Protein Kinases

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially inToxoplasma gondiiwhere 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division inT. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growthin vitroor infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes inT. gondii.

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Beskrywing, modellering en dok-studies van Plasmodium falciparum kinase PfCDPK4

Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie ◽

10.36303/satnt.2019.38.1.677 ◽

2019 ◽

pp. 26-40

Author(s):

Thomas Makungo ◽

Tsepo Tsekoa ◽

Anjo Theron ◽

Dalu Mancama ◽

Teunis van Ree

Keyword(s):

Plasmodium Falciparum ◽

Protein Kinases ◽

In Silico ◽

Calcium Dependent ◽

Dependent Protein ◽

Calcium Dependent Protein Kinases

Met die toenemende voorkoms van weerstandige Plasmodium stamme het die beheer van malaria-voorkoms en -mortaliteit weer op die voorgrond getree. Nuwe teikens en antimalariamiddels wat effektief is teen weerstandige malaria-parasiete word dus dringend benodig. Kalsium-afhanklike proteïenkinases (calcium dependent protein kinases – CDPKs) is betrokke by die beheer van ’n aantal biologiese prosesse in die malaria-parasiet, Plasmodium falciparum, met CDPK4 die belangrikste ensiem in hierdie klas. In hierdie studie is die struktuur van PfCDPK4 gebruik as templaat vir die soeke na nuwe malariamiddels. Die PfCDPK4 modelstruktuur is deur middel van homologiemodellering gegenereer en die stereochemiese kwaliteit gevalideer. Die molekulêre modelleringbenadering deur middel van in silico sifting teen die teiken-molekuul PfCDPK4 het ’n beskeie biblioteek van 20 000 chemiese verbindings ingesluit, asook ’n aantal aktiewe natuurprodukte en kliniesgoedgekeurde kinase-inhibeerders. In silico sifting van die Biofocus biblioteek teen PfCDPK4 het 26 verbindings opgelewer; in vitro sifting het bevestig dat drie van hierdie verbindings matig aktief is teen Plasmodium falciparum NF54, met persentasie inhibisie tussen 42% en 47%.

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