Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells

Autophagy Protein Atg3 is Essential for Maintaining Mitochondrial Integrity and for Normal Intracellular Development of Toxoplasma gondii Tachyzoites

Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites

Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome-lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3(+) structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival

A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle

Observational study of the medical management of patients with peripheral artery disease.

Background Previous research has suggested that patients with peripheral artery disease (PAD) are not offered adequate risk factor modification, despite their high cardiovascular risk. The aim of this study was to assess the cardiovascular profiles of patients with PAD and quantify the survival benefits of target-based risk factor modification. Methods The Vascular and Endovascular Research Network (VERN) prospectively collected cardiovascular profiles of patients with PAD from ten UK vascular centres (April to June 2018) to assess practice against UK and European goal-directed best medical therapy guidelines. Risk and benefits of risk factor control were estimated using the SMART-REACH model, a validated cardiovascular prediction tool for patients with PAD. Results Some 440 patients (mean(s.d.) age 70(11) years, 24·8 per cent women) were included in the study. Mean(s.d.) cholesterol (4·3(1·2) mmol/l) and LDL-cholesterol (2·7(1·1) mmol/l) levels were above recommended targets; 319 patients (72·5 per cent) were hypertensive and 343 (78·0 per cent) were active smokers. Only 11·1 per cent of patients were prescribed high-dose statin therapy and 39·1 per cent an antithrombotic agent. The median calculated risk of a major cardiovascular event over 10 years was 53 (i.q.r. 44–62) per cent. Controlling all modifiable cardiovascular risk factors based on UK and European guidance targets (LDL-cholesterol less than 2 mmol/l, systolic BP under 140 mmHg, smoking cessation, antiplatelet therapy) would lead to an absolute risk reduction of the median 10-year cardiovascular risk by 29 (20–38) per cent with 6·3 (4·0–9·3) cardiovascular disease-free years gained. Conclusion The medical management of patients with PAD in this secondary care cohort was suboptimal. Controlling modifiable risk factors to guideline-based targets would confer significant patient benefit

Observational study of the medical management of patients with peripheral artery disease

Background: Previous research has suggested that patients with peripheral artery disease (PAD) are not offered adequate risk factor modification, despite their high cardiovascular risk. The aim of this study was to assess the cardiovascular profiles of patients with PAD and quantify the survival benefits of target-based risk factor modification. Methods: The Vascular and Endovascular Research Network (VERN) prospectively collected cardiovascular profiles of patients with PAD from ten UK vascular centres (April to June 2018) to assess practice against UK and European goal-directed best medical therapy guidelines. Risk and benefits of risk factor control were estimated using the SMART-REACH model, a validated cardiovascular prediction tool for patients with PAD. Results: Some 440 patients (mean(s.d.) age 70(11) years, 24·8 per cent women) were included in the study. Mean(s.d.) cholesterol (4·3(1·2) mmol/l) and LDL-cholesterol (2·7(1·1) mmol/l) levels were above recommended targets; 319 patients (72·5 per cent) were hypertensive and 343 (78·0 per cent) were active smokers. Only 11·1 per cent of patients were prescribed high-dose statin therapy and 39·1 per cent an antithrombotic agent. The median calculated risk of a major cardiovascular event over 10 years was 53 (i.q.r. 44–62) per cent. Controlling all modifiable cardiovascular risk factors based on UK and European guidance targets (LDL-cholesterol less than 2 mmol/l, systolic BP under 140 mmHg, smoking cessation, antiplatelet therapy) would lead to an absolute risk reduction of the median 10-year cardiovascular risk by 29 (20–38) per cent with 6·3 (4·0–9·3) cardiovascular disease-free years gained. Conclusion: The medical management of patients with PAD in this secondary care cohort was suboptimal. Controlling modifiable risk factors to guideline-based targets would confer significant patient benefit