Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

Alam, Mahmood M.; Solyakov, Lev; Bottrill, Andrew R.; Flueck, Christian; Siddiqui, Faiza Amber; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate D; Hopp, Christine S.; Chitnis, Chetan E.; Doerig, Christian; Moon, Robert W.; Green, Judith L.; Holder, Anthony A.; Baker, David A.; Tobin, Andrew B.

doi:10.1038/ncomms8285

Cited by 171 publications

(234 citation statements)

References 63 publications

Supporting

Mentioning

226

Contrasting

Order By: Relevance

“…Although both TgCDPK1 and TgCDPK3 regulate egress in T. gondii, this function is provided by CDPK5 in P. falciparum (25). In both systems, protein kinase G (PKG) is required for egress (23,25), and invasion (26,27), demonstrating that cyclic nucleotide-and calcium-dependent kinase pathways are intertwined. Although functional studies have addressed the roles of many canonical CDPKs, much less is known about those with noncanonical domain structures.…”

Section: -Isoleucine [I]-lysine [K]-lysine [K]) Kinases In Plasmodiummentioning

confidence: 99%

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

2016

View full text Add to dashboard Cite

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 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 in T. 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 (CRISPRassociated 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 growth in vitro or 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 in T. gondii.

show abstract

Section: -Isoleucine [I]-lysine [K]-lysine [K]) Kinases In Plasmodiummentioning

confidence: 99%

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

2016

View full text Add to dashboard Cite

show abstract

“…This was carried out as previously described [28] with one difference in that peptides were eluted from a reverse-phase PicoFrit capillary column (75 µm i.d. × 400 mm) over a period of 2 h.…”

Section: Methodsmentioning

confidence: 99%

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Mitcheson

Bottrill

Carr

et al. 2016

Malar J

Self Cite

View full text Add to dashboard Cite

BackgroundExamining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite.ResultsIntroduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2.ConclusionsAllelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2.

show abstract

“…Initially, a plasmodial guanylyl cyclase becomes activated, leading to the synthesis of cyclic GMP (cGMP) (Carucci et al, 2000;Muhia et al, 2001). The rise of cGMP activates the cGMP-dependent protein kinase G, which regulates the generation of PIP 2 (Alam et al, 2015;Brochet et al, 2014;McRobert et al, 2008). At the same time, PI-PLC is stimulated, which hydrolyses PIP 2 to generate DAG and IP 3 (Martin et al, 1994;Raabe et al, 2011b), resulting in the release of calcium from internal stores (Billker et al, 2004).…”

Section: The Role Of Plasmodial Phospholipases During Life-cycle Progmentioning

confidence: 99%

Phospholipases during membrane dynamics in malaria parasites

Flammersfeld

Lang

Flieger

et al. 2018

International Journal of Medical Microbiology

View full text Add to dashboard Cite

A B S T R A C TPlasmodium parasites, the causative agents of malaria, display a well-regulated lipid metabolism required to ensure their survival in the human host as well as in the mosquito vector. The fine-tuning of lipid metabolic pathways is particularly important for the parasites during the rapid erythrocytic infection cycles, and thus enzymes involved in lipid metabolic processes represent prime targets for malaria chemotherapeutics. While plasmodial enzymes involved in lipid synthesis and acquisition have been studied in the past, to date not much is known about the roles of phospholipases for proliferation and transmission of the malaria parasite. These phospholipid-hydrolyzing esterases are crucial for membrane dynamics during host cell infection and egress by the parasite as well as for replication and cell signaling, and thus they are considered important virulence factors. In this review, we provide a comprehensive bioinformatic analysis of plasmodial phospholipases identified to date. We further summarize previous findings on the lipid metabolism of Plasmodium, highlight the roles of phospholipases during parasite life-cycle progression, and discuss the plasmodial phospholipases as potential targets for malaria therapy.

show abstract

Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

Cited by 171 publications

References 63 publications

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

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

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Phospholipases during membrane dynamics in malaria parasites

Contact Info

Product

Resources

About