Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility.

IMPORTANCE: This work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium-dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating that it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive kinase alleles and conditionally depleted alleles uncovered epistasis between CDPK2A and CDPK1, implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting kinases for anti-parasitic therapies.

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress.

Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, we assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with a delayed phenotype, was shown to be a regulator of invasion and egress. We named the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology.

A PEROXO-Tag Enables Rapid Isolation of Peroxisomes from Human Cells.

Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than 1 h to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally localized 3XHA epitope tag ("PEROXO-Tag") and associated immunoprecipitation ("PEROXO-IP") workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 min post homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes.

Plate-Based Quantification of Stimulated Toxoplasma Egress.

Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by which apicomplexan parasites like Toxoplasma gondii exit from host cells, rupturing the parasitophorous vacuole and host-cell plasma membranes in the process. T. gondii retains the ability to egress throughout most of its intracellular replicative cycle, and this process has been associated with parasite signaling pathways that include the modulation of intracellular calcium, cyclic nucleotides, phosphatidic acid, and pH, which can be manipulated genetically or pharmacologically. Here we describe two methods of assessing stimulated parasite egress from host cells by measuring the permeabilization of host-cell membranes that occurs during this process. The first method measures the release of lactate dehydrogenase (LDH) from host cells, which is quantified in a colorimetric assay that detects LDH by the enzymatic generation of red formazan. The second method measures entry of the cell-impermeant 4',6-diamidino-2-phenylindole (DAPI) DNA dye, which stains host-cell nuclei (HCN) as parasites egress. Both described methods complement, with higher throughput, video-microscopy approaches that are well suited to examine the dissociation of parasite vacuoles that follows host-cell permeabilization.

Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii.

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depolymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites.

A qualitative study of infant feeding decisions among low-income women in the Republic of Ireland.

OBJECTIVE: to explore infant feeding decisions among low-income women living in Ireland to gain an in-depth understanding of the factors, which influence breast feeding initiation and continuation. DESIGN: a descriptive qualitative study using focus groups and semi-structured interviews. SETTING: community and primary health-care settings in the Republic of Ireland. PARTICIPANTS: a convenience sample of 33 low-income mothers was recruited from 2 community programmes and 3 primary health-care centres. FINDINGS: six dominant themes were identified using Thematic Analysis. Prior knowledge of infant feeding, especially from experiences of seeing breast- and artificial milk-feeding in the family and the community, influenced feeding choice. Embarrassment and stigma about breast feeding in public places and in some cases in the private sphere were commonly described as barriers to breast feeding. The decision to bottle feed often reflected a balancing of the needs of the mother and the baby, because breast feeding was often perceived as inconvenient and requiring extreme determination. Breast feeding difficulties in the early weeks were frequently described and those who stopped breast feeding early often lacked practical knowledge and experienced support. In terms of health professional support, the mothers favoured a non-pressurised approach along with practical help with breast feeding. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: there is a need for promotional efforts to normalise breast feeding and for training of health professionals in the provision of appropriate support.