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The coccidian subgroup of Apicomplexa possesses an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR). The APR serves as microtubule-organizing center for the 22 subpellicular microtubules (SPMTs) that are linked to a patchwork of flattened vesicles, via an intricate network composed of alveolins. Here, we capitalize on ultrastructure expansion microscopy (U-ExM) to localize the Toxoplasma gondii Apical Cap protein 9 (AC9) and its partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Parasites conditionally depleted in AC9 or AC10 replicate normally but are defective in microneme secretion and fail to invade and egress from infected cells. Electron microscopy revealed that the mature parasite mutants are conoidless, while U-ExM highlighted the disorganization of the SPMTs which likely results in the catastrophic loss of APR and conoid.

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

The obligate intracellular parasite Toxoplasma gondii possesses a repertoire of 11 myosins. Three class XIV motors participate in motility, invasion and egress, whereas the class XXII myosin F is implicated in organelle positioning and inheritance of the apicoplast. Here we provide evidence that TgUNC acts as a chaperone dedicated to the folding, assembly and function of all Toxoplasma myosins. The conditional ablation of TgUNC recapitulates the phenome of the known myosins and uncovers two functions in parasite basal complex constriction and synchronized division within the parasitophorous vacuole. We identify myosin J and centrin 2 as essential for the constriction. We demonstrate the existence of an intravacuolar cell–cell communication ensuring synchronized division, a process dependent on myosin I. This connectivity contributes to the delayed death phenotype resulting from loss of the apicoplast. Cell–cell communication is lost in activated macrophages and during bradyzoite differentiation resulting in asynchronized, slow division in the cysts. The mechanism by which Toxoplasma gondii achieves synchronized cell division is incompletely understood. Here, the authors identify an intravacuolar cell-cell communication that ensures synchronized division and depends on myosin I.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient’s isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization. Köhler et al. document the treatment, and clinical improvement, of a male patient with a life-threatening chronic multi-drug resistant Pseudomonas aeruginosa infection with aerosolized personalized phage therapy, in combination with intravenous antibiotic treatment.

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface. Host cell invasion by Toxoplasma gondii depends on the heavily phosphorylated RON complex, but the relevance and regulation of these modifications are not understood. Here, the authors identify the kinase RON13 as a key virulence factor, determine its structure and show that it phosphorylates the RON complex.

This study has used dense reconstructions from serial EM images to compare the neuropil ultrastructure and connectivity of aged and adult mice. The analysis used models of axons, dendrites, and their synaptic connections, reconstructed from volumes of neuropil imaged in layer 1 of the somatosensory cortex. This shows the changes to neuropil structure that accompany a general loss of synapses in a well-defined brain region. The loss of excitatory synapses was balanced by an increase in their size such that the total amount of synaptic surface, per unit length of axon, and per unit volume of neuropil, stayed the same. There was also a greater reduction of inhibitory synapses than excitatory, particularly those found on dendritic spines, resulting in an increase in the excitatory/inhibitory balance. The close correlations, that exist in young and adult neurons, between spine volume, bouton volume, synaptic size, and docked vesicle numbers are all preserved during aging. These comparisons display features that indicate a reduced plasticity of cortical circuits, with fewer, more transient, connections, but nevertheless an enhancement of the remaining connectivity that compensates for a generalized synapse loss.

The apicoplast, a relic plastid organelle derived from secondary endosymbiosis, is crucial for many medically relevant Apicomplexa. While it no longer performs photosynthesis, the organelle retains several essential metabolic pathways. In this study, we examine the four primary metabolic pathways in the Toxoplasma gondii apicoplast, along with an accessory pathway, and identify conditions that can bypass these. Contrary to the prevailing view that the apicoplast is indispensable for T. gondii , we demonstrate that bypassing all pathways renders the apicoplast non-essential. We further show that T. gondii lacking an apicoplast ( T. gondii −Apico ) can be maintained indefinitely in culture, establishing a unique model to study the functions of this organelle. Through comprehensive metabolomic, transcriptomic, and proteomic analyses of T. gondii −Apico we uncover significant adaptation mechanisms following loss of the organelle and identify numerous putative apicoplast proteins revealed by their decreased abundance in T. gondii −Apico . Moreover, T. gondii −Apico parasites exhibit reduced sensitivity to apicoplast targeting compounds, providing a valuable tool for discovering new drugs acting on the organelle. The capability to culture T. gondii without its plastid offers new avenues for exploring apicoplast biology and developing novel therapeutic strategies against apicomplexan parasites. Here, Chen et al. bypass the critical metabolic pathways of the remnant plastid organelle of Toxoplasma gondii . A subsequent detailed characterization of parasites devoid of the apicoplast organelle provides unprecedented insights into its constituents, evolution and functions, uncovering new vulnerabilities.

Rhoptries are specialized secretory organelles conserved across the Apicomplexa phylum, essential for host cell invasion and critical for subverting of host cellular and immune functions. They contain proteins and membranous materials injected directly into the host cells, participating in parasitophorous vacuole formation. Toxoplasma gondii tachyzoites harbor 8 to 12 rhoptries, 2 of which are docked to an apical vesicle (AV), a central element associated with a rhoptry secretory apparatus prior to injection into the host cell. This parasite is also equipped with 5 to 6 microtubule-associated vesicles, presumably serving as AV replenishment for iterative rhoptry discharge. Here, we characterized a rhoptry protein, rhoptry discharge factor 3 (RDF3), crucial for rhoptry discharge and invasion. RDF3 enters the secretory pathway, localizing near the AV and associated with the rhoptry bulb. Upon invasion, RDF3 dynamically delocalizes, suggesting a critical role at the time of rhoptry discharge. Cryo-electron tomography analysis of RDF3-depleted parasites reveals irregularity in microtubule-associated vesicles morphology, presumably impacting on their preparedness to function as an AV. Our findings suggest that RDF3 is priming the microtubule-associated vesicles for rhoptry discharge by a mechanism distinct from the rhoptry secretory apparatus contribution.

In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei . This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum . CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites. Despite functional optimisation during evolution of parasitism, most members of a calcium dependent protein kinase (CDPK) family show genetic redundancy in Plasmodium . Here, the authors screen 294 genetic interactions among protein kinases in Plasmodium and show how some CDPKs functionally interact to control motility and host cell invasion.

In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10–12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity. The authors identified a series of cytoskeletal proteins involved in the discharge of invasion-related organelles in Toxoplasma gondii . They successfully delineated their functions through the utilization of expansion and cryo-electron microscopy.