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Toxoplasma gondii is a protozoan apicomplexan parasite that uses an adhesion-dependent mode of motility termed gliding to access host cells and disseminate into tissues. Previous studies on Apicomplexa motile morphotypes, including the T. gondii tachyzoite, have identified a cortical actin-myosin motor system that drives the rearward translocation of transmembrane adhesins, thus powering forward movement. However, this model is currently questioned. Here, combining micropatterning and tunable surface chemistry (to edit parasite surface ligands) with flow force and live or super-resolution imaging, we show that tachyzoites build only one apical anchoring contact with the substrate, over which it slides. Furthermore, we show that glycosaminoglycan-parasite interactions are sufficient to promote such force-productive contact and find that the apicobasal flow is set up independent of adhesin release and surface interactions. These findings should enable further characterization of the molecular functions at the T. gondii-substrate mechanosensitive interface and their comparison across apicomplexans., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The functional role of platelets is intricately linked to the dynamic organization of two main components of the cytoskeleton, microtubules and actin fibers. Throughout the phases of platelet activation, spreading, and retraction, both of these essential polymers undergo continuous and orchestrated reorganization. Our investigation of the dynamic cytoskeletal changes during these phases highlights a sequential remodeling of the actin cytoskeleton in adherent platelets from the formation of initial actin nodules through the development of stress fibers and a subsequent return to nodular structures. Concurrently, the marginal ring of microtubules, characteristic of resting platelets, undergoes a re-organization induced by marginal band extension and coiling toward the formation of star-like bundles of microtubules. Subsequently, these bundles are dispersed into individual microtubules, which are re-bundled at later stages before ring-like structures are formed again. These findings suggest a compelling tendency for both cytoskeletal components to revert to their original configurations. Notably, the early steps of platelet cytoskeleton reorganizations have previously been shown to be regulated by the signaling cascade triggered during platelet activation, which leads to an increase of cytosolic calcium concentrations. We show here that later steps are potentially regulated by a progressive decrease of intracellular calcium concentrations as platelets approach the end of their functional lifespan.

The French Society for Cell Biology (SBCF) is actively involved in communicating the latest advances and organizing scientific events, as well as supporting young researchers, in this field. The SBCF also supports and organizes outreaching activities designed to raise public awareness of science in general and cell biology in particular. The Society, in its present form, was founded in 1984. To mark this milestone, we are organizing a memorable symposium hosted by the Académie des Sciences (https://sbcf.fr/en/event/symposium-des-40-ans-de-la-sbcf/) on September 10, 2024., (© 2024 The Author(s). Biology of the Cell published by Wiley‐VCH GmbH on behalf of Société Française des Microscopies and Société de Biologie Cellulaire de France.)

The protozoa, Toxoplasma gondii and Plasmodium spp., are preeminent members of the Apicomplexa parasitic phylum in large part due to their public health and economic impact. Hence, they serve as model unicellular eukaryotes with which to explore the repertoire of molecular and cellular strategies that specific developmental morphotypes deploy to timely adjust to their host(s) in order to perpetuate. In particular, host tissue- and cell-invasive morphotypes termed zoites alternate extracellular and intracellular lifestyles, thereby sensing and reacting to a wealth of host-derived biomechanical cues over their partnership. In the recent years, biophysical tools especially related to real time force measurement have been introduced, teaching us how creative are these microbes to shape a unique motility system that powers fast gliding through a variety of extracellular matrices, across cellular barriers, in vascular systems or into host cells. Equally performant was this toolkit to start illuminating how parasites manipulate their hosting cell adhesive and rheological properties to their advantage. In this review, besides highlighting major discoveries along the way, we discuss the most promising development, synergy, and multimodal integration in active noninvasive force microscopy methods. These should in the near future unlock current limitations and allow capturing, from molecules to tissues, the many biomechanical and biophysical interplays over the dynamic host and microbe partnership., (© 2023 The Authors. Biology of the Cell published by Wiley-VCH GmbH on behalf of Société Française des Microscopies and Société de Biologie Cellulaire de France.)

The Toxoplasma gondii tachyzoite is a singled-cell obligate intracellular parasite responsible for the acute phase of toxoplasmosis. This polarized cell exhibits an apical complex, a hallmark of the phylum Apicomplexa, essential for motility, invasion, and egress from the host cell. Located on the opposite end of the cell is the basal complex, an elaborated cytoskeletal structure that also plays critical roles in the lytic cycle of the parasite, being involved in motility, cell division, constriction and cytokinesis, as well as intravacuolar cell-cell communication. Nevertheless, only a few proteins of this structure have been described and functionally assessed. In this study, we used spatial proteomics to identify new basal complex components (BCC), and in situ imaging, including ultrastructure expansion microscopy, to position them. We thus confirmed the localization of nine BCCs out of the 12 selected candidates and assigned them to different sub-compartments of the basal complex, including two new domains located above the basal ring and below the posterior cup. Their functional investigation revealed that none of these BCCs are essential for parasite growth in vitro . However, one BCC is critical for constricting of the basal complex, likely through direct interaction with the class VI myosin heavy chain J (MyoJ), and for gliding motility. Four other BCCs, including a phosphatase and a guanylate-binding protein, are involved in the formation and/or maintenance of the intravacuolar parasite connection, which is required for the rosette organization and synchronicity of cell division., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roumégous, Abou Hammoud, Fuster, Dupuy, Blancard, Salin, Robinson, Renesto, Tardieux and Frénal.)

T. gondii is a eukaryotic parasite that has evolved a stage called tachyzoite which multiplies in host cells by producing two daughter cells internally. These nascent tachyzoites bud off their mother and repeat the division process until the expanding progenies escape to settle and multiply in other host cells. Over these intra- and extra-cellular phases, the tachyzoite maintains an essential apicobasal polarity that emerges through a unique bidirectional budding process of the elongating cells. This process requires the assembly of several molecular complexes that, at the nascent pole, encompass structural and myosin motor elements. To characterize a recently identified basal pole marker named BCC7 with respect to the posterior myosin J and myosin C motors, we used conventional biochemistry as well as advanced proteomic and in silico analysis in conjunction with live and super resolution microscopy of transgenic fluorescent tachyzoites. We document that BCC7 forms a ribbed ring below which myosin C motor entities distribute regularly. In addition, we identified-among 13 BCC7 putative partners-two novel and five known members of the inner membrane complex (IMC) family which ends at the apical side of the ring. Therefore, BCC7 could assist the stabilization of the IMC plaques and contribute to the parasite biomechanical properties.

The intracellular protozoan parasites of the Leishmania genus are responsible for Leishmaniases, vector borne diseases with a wide range of clinical manifestations. Leishmania (L.) donovani causes visceral leishmaniasis (kala azar), the most severe of these diseases. Along their biological cycle, Leishmania parasites undergo distinct developmental transitions including metacyclogenesis and differentiation of metacyclic promastigotes (MPs) to amastigotes. Metacyclogenesis inside the phlebotomine sandfly host's midgut converts the procyclic dividing promastigotes to non-dividing infective MPs eventually injected into the skin of mammalian hosts and phagocytosed by macrophages where the MPs are converted inside modified phagolysosomes to the intracellular amastigotes. These developmental transitions involve dramatic changes in cell size and shape and reformatting of the flagellum requiring thus membrane and cytoskeleton remodeling in which phosphoinositide (PI) signaling and metabolism must play central roles. This study reports on the LDBPK_220120.1 gene, the L. donovani ortholog of LmjF.22.0250 from L. major that encodes a phosphatase from the "Atypical Lipid Phosphatases" (ALPs) enzyme family. We confirmed the expression of the LDBPK_220120.1 gene product in both L. donovani promastigotes and axenic amastigotes and showed that it behaves in vitro as a Dual Specificity P-Tyr and monophosphorylated [PI(3)P and PI(4)P] PI phosphatase and therefore named it Ld TyrPIP_22 (L eishmaniad onovani Tyrosine PI Phosphatase, gene locus at chromosome 22). By immunofluorescence confocal microscopy we localized the Ld TyrPIP_22 in several intracellular sites in the cell body of L. donovani promastigotes and amastigotes and in the flagellum. A temperature and pH shift from 25°C to 37°C and from pH 7 to 5.5, induced a pronounced recruitment of Ld TyrPIP_22 epitopes to the flagellar pocket and a redistribution around the nucleus. These results suggest possible role(s) for this P-Tyr/PI phosphatase in the regulation of processes initiated or upregulated by this temperature/pH shift that contribute to the developmental transition from MPs to amastigotes inside the mammalian host macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Papadaki, Tziouvara, Kotopouli, Koumarianou, Doukas, Rios, Tardieux, Köhn and Boleti.)

Background: Biomarker discovery remains a major challenge for predictive medicine, in particular, in the context of chronic diseases. This is true for the widespread protozoan Toxoplasma gondii which establishes long-lasting parasitism in metazoans, humans included. This microbe successively unfolds distinct genetic programs that direct the transition from high to low replicative potential inside host cells. As a slow-replicating cell, the T. gondii bradyzoite developmental stage persists enclosed in a cyst compartment within tissues including the nervous system, being held by a sustained immune equilibrium which accounts for the prolonged clinically silent phase of parasitism. Serological surveys indicate that nearly one third of the human population has been exposed to T. gondii and possibly host bradyzoites. Because any disruption of the immune balance drives the reverse transition from bradyzoite to fast replicating tachyzoite and uncontrolled growth of the latter, these people are at risk for life-threatening disease. While serological tests for discriminating recent from past infection are available, there is yet no immunogenic biomarker used in the serological test to allow ascertaining the presence of persistent bradyzoites., Results: Capitalizing on genetically engineered parasites induced to produce mature bradyzoites in vitro, we have identified the BCLA/MAG2 protein being restricted to the bradyzoite and the cyst envelope. Using laboratory mice as relevant T. gondii host models, we demonstrated that BCLA/MAG2 drives the generation of antibodies that recognize bradyzoite and the enveloping cyst structure. We have designed an ELISA assay based on a bacterially produced BCLA recombinant polypeptide, which was validated using a large collection of sera from mice of different genetic backgrounds and infected with bcla+ or bcla-null cystogenic and non-cystogenic T. gondii strains. To refine the design of the ELISA assay, we applied high-resolution BCLA epitope mapping and identified a specific combination of peptides and accordingly set up a selective and sensitive ELISA assay which allowed the detection of anti-BCLA/MAG2 antibodies in the sera of human patients with various forms of toxoplasmosis., Conclusions: We brought proof of principle that anti-BCLA/MAG2 antibodies serve as specific and sensitive serological markers in the perspective of a combinatorial strategy for detection of persistent T. gondii parasitism.

N -myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N -myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Myristoylation often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence suggests additional regulatory roles for myristoylation on proteins. Here we describe the myristoylated proteome of Toxoplasma gondii using chemoproteomic methods and show that a small-molecule NMT inhibitor developed against related Plasmodium spp . is also functional in Toxoplasma . We identify myristoylation on a transmembrane protein, the microneme protein 7 (MIC7), which enters the secretory pathway in an unconventional fashion with the myristoylated N-terminus facing the lumen of the micronemes. MIC7 and its myristoylation play a crucial role in the initial steps of invasion, likely during the interaction with and penetration of the host cell. Myristoylation of secreted eukaryotic proteins represents a substantial expansion of the functional repertoire of this co-translational modification., Competing Interests: MB, CD, LV, SN, EB, BT, AH, BW, SF, SM, JY, IT, MT No competing interests declared, ET EWT is a founder, shareholder and Director of Myricx Pharma Ltd, (© 2020, Broncel et al.)

Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the Plasmodium sporozoite and the Toxoplasma tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two parasites and is largely characterized. Localized beneath the cell surface, it includes actin filaments, unconventional myosin motors housed within a multimember glideosome unit, and apically secreted transmembrane adhesins. In contrast, less is known about the force mechanisms powering cell movement. Pioneered biophysical studies on the sporozoite and phenotypic analysis of tachyzoite actin-related mutants have added complexity to the general view that force production for parasite forward movement directly results from the myosin-driven rearward motion of the actin-coupled adhesion sites. Here, we have interrogated how forces and substrate adhesion-de-adhesion cycles operate and coordinate to allow the typical left-handed helical gliding mode of the tachyzoite. By combining quantitative traction force and reflection interference microscopy with micropatterning and expansion microscopy, we unveil at the millisecond and nanometer scales the integration of a critical apical anchoring adhesion with specific traction and spring-like forces. We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital helical gliding capacity.

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells., Competing Interests: The authors have declared that no competing interests exist.

Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems., Competing Interests: The authors have declared that no competing interests exist.

Host cell invasion by Toxoplasma gondii/T. gondii tachyzoites is an obligate but complex multistep process occurring in second-scale. To capture the dynamic nature of the whole entry process requires fast and high-resolution live cell imaging. Recent advances in T. gondii/host cell genome editing and in quantitative live cell imaging-image acquisition and processing included-provide a systematic way to accurately phenotype T. gondii tachyzoite invasive behaviour and to highlight any variation or default from a standard scenario. Therefore, applying these combined strategies allows gaining deeper insights into the complex mechanisms underlying host cell invasion.

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites., (© 2019 The Author. Published under the terms of the CC BY 4.0 license.)

Intracellular protozoans co-evolved with their mammalian host cells a range of strategies to cope with the composite and dynamic cell surface features they encounter during migration and infection. Therefore, these single-celled eukaryotic parasites represent a fascinating source of living probes for precisely capturing the dynamic coupling between the membrane and contractile cortex components of the cell surface. Such biomechanical changes drive a constant re-sculpting of the host cell surface, enabling rapid adjustments that contribute to cellular homeostasis. As emphasized in this review, through the design of specific molecular devices and stratagems to interfere with the biomechanics of the mammalian cell surface these parasitic microbes escape from dangerous or unfavourable microenvironments by breaching host cell membranes, directing the membrane repair machinery to wounded membrane areas, or minimizing membrane assault using discretion and speed when invading host cells for sustained residence., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The protozoan parasite Toxoplasma gondii has co-evolved with its homeothermic hosts (humans included) strategies that drive its quasi-asymptomatic persistence in hosts, hence optimizing the chance of transmission to new hosts. Persistence, which starts with a small subset of parasites that escape host immune killing and colonize the so-called immune privileged tissues where they differentiate into a low replicating stage, is driven by the interleukin 12 (IL-12)-interferon-γ (IFN-γ) axis. Recent characterization of a family of Toxoplasma effectors that are delivered into the host cell, in which they rewire the host cell gene expression, has allowed the identification of regulators of the IL-12-IFN-γ axis, including repressors. We now report on the dense granule-resident effector, called TEEGR (Toxoplasma E2F4-associated EZH2-inducing gene regulator) that counteracts the nuclear factor-κB (NF-κB) signalling pathway. Once exported into the host cell, TEEGR ends up in the nucleus where it not only complexes with the E2F3 and E2F4 host transcription factors to induce gene expression, but also promotes shaping of a non-permissive chromatin through its capacity to switch on EZH2. Remarkably, EZH2 fosters the epigenetic silencing of a subset of NF-κB-regulated cytokines, thereby strongly contributing to the host immune equilibrium that influences the host immune response and promotes parasite persistence in mice.

The intracellular parasite Toxoplasma gondii, hijacks evolutionarily conserved host processes by delivering effector proteins into the host cell that shift gene expression in a timely fashion. We identified a parasite dense granule protein as GRA18 that once released in the host cell cytoplasm forms versatile complexes with regulatory elements of the β-catenin destruction complex. By interacting with GSK3/PP2A-B56, GRA18 drives β-catenin up-regulation and the downstream effects on host cell gene expression. In the context of macrophages infection, GRA18 induces the expression of a specific set of genes commonly associated with an anti-inflammatory response that includes those encoding chemokines CCL17 and CCL22. Overall, this study adds another original strategy by which T. gondii tachyzoites reshuffle the host cell interactome through a GSK3/β-catenin axis to selectively reprogram immune gene expression., Competing Interests: HH, MB, LB, AK, BT, YC, IT, MH, AB No competing interests declared, (© 2018, He et al.)

Dinitroanilines are chemical compounds with high selectivity for plant cell α-tubulin in which they promote microtubule depolymerization. They target α-tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum , which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum . These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.

To invade cells, the parasite Toxoplasma gondii injects a multi-unit nanodevice into the target cell plasma membrane (PM). The core nanodevice, which is composed of the RhOptry Neck (RON) protein complex, connects Toxoplasma and host cell through a circular tight junction (TJ). We now report that this RON nanodevice mechanically promotes membrane scission at the TJ-PM interface, directing a physical rotation driven by the parasite twisting motion that enables the budding parasitophorous vacuole (PV) to seal and separate from the host cell PM as a bona fide subcellular Toxoplasma-loaded PV. Mechanically impairing the process induces swelling of the budding PV and death of the parasite but not host cell. Moreover, this study reveals that the parasite nanodevice functions as a molecular trigger to promote PV membrane remodeling and rapid onset of T. gondii to intracellular lifestyle., (Copyright © 2018 Elsevier Inc. All rights reserved.)

An unusual genome architecture characterizes the two related human parasitic pathogens Plasmodium falciparum and Toxoplasma gondii. A major fraction of the bulk parasite genome is packaged as transcriptionally permissive euchromatin with few loci embedded in silenced heterochromatin. Primary chromatin shapers include histone modifications at the nucleosome lateral surface close to the DNA but their mode of action remains unclear. We now identify versatile modifications at Lys31 within the globular domain of histone H4 that crucially determine genome organization and expression in Apicomplexa parasites. H4K31 acetylation at the promoter correlates with, and perhaps directly regulates, gene expression in both parasites. By contrast, monomethylated H4K31 is enriched in the core body of T. gondii active genes but inversely correlates with transcription, whereas it is unexpectedly enriched at transcriptionally inactive pericentromeric heterochromatin in P. falciparum , a region devoid of the characteristic H3K9me3 histone mark and its downstream effector HP1.

While the intracellular parasite Toxoplasma relies on a divergent actomyosin motor to support unique speeds in directional movement, the dynamics and architecture of parasite actin filaments remain a much-discussed issue. Using actin chromobodies, Periz et al. started to unveil how networks of dynamic F-actin connect Toxoplasma progeny and expand in the replicative vacuole., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The several-micrometer-sized Toxoplasma gondii protozoan parasite invades virtually any type of nucleated cell from a warm-blooded animal within seconds. Toxoplasma initiates the formation of a tight ring-like junction bridging its apical pole with the host cell membrane. The parasite then actively moves through the junction into a host cell plasma membrane invagination that delineates a nascent vacuole. Recent high resolution imaging and kinematics analysis showed that the host cell cortical actin dynamics occurs at the site of entry while gene silencing approaches allowed motor-deficient parasites to be generated, and suggested that the host cell could contribute energetically to invasion. In this study we further investigate this possibility by analyzing the behavior of parasites genetically impaired in different motor components, and discuss how the uncovered mechanisms illuminate our current understanding of the invasion process by motor-competent parasites., Results: By simultaneously tracking host cell membrane and cortex dynamics at the site of interaction with myosin A-deficient Toxoplasma, the junction assembly step could be decoupled from the engagement of the Toxoplasma invasive force. Kinematics combined with functional analysis revealed that myosin A-deficient Toxoplasma had a distinct host cell-dependent mode of entry when compared to wild-type or myosin B/C-deficient Toxoplasma. Following the junction assembly step, the host cell formed actin-driven membrane protrusions that surrounded the myosin A-deficient mutant and drove it through the junction into a typical vacuole. However, this parasite-entry mode appeared suboptimal, with about 40 % abortive events for which the host cell membrane expansions failed to cover the parasite body and instead could apply deleterious compressive forces on the apical pole of the zoite., Conclusions: This study not only clarifies the key contribution of T. gondii tachyzoite myosin A to the invasive force, but it also highlights a new mode of entry for intracellular microbes that shares early features of macropinocytosis. Given the harmful potential of the host cell compressive forces, we propose to consider host cell invasion by zoites as a balanced combination between host cell membrane dynamics and the Toxoplasma motor function. In this light, evolutionary shaping of myosin A with fast motor activity could have contributed to optimize the invasive potential of Toxoplasma tachyzoites and thereby their fitness.

The capacity to migrate is fundamental to multicellular and single-celled life. Apicomplexan parasites, an ancient protozoan clade that includes malaria parasites (Plasmodium) and Toxoplasma, achieve remarkable speeds of directional cell movement. This rapidity is achieved via a divergent actomyosin motor system, housed within a narrow compartment that lies underneath the length of the parasite plasma membrane. How this motor functions at a mechanistic level during motility and host cell invasion is a matter of debate. Here, we integrate old and new insights toward refining the current model for the function of this motor with the aim of revitalizing interest in the mechanics of how these deadly pathogens move., (© 2016 Tardieux and Baum.)

An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism., (© 2016 Gay et al.)

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera, Plasmodium and Toxoplasma, until we genetically engineered viable parasites lacking AMA1. The reduction in invasiveness of the Toxoplasma gondii RH-AMA1 knockout (RH-AMA1(KO)) tachyzoite population, in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1(KO) tachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1(KO) population amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type II T. gondii genotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite population in vivo., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The public health threats imposed by toxoplasmosis worldwide and by malaria in sub-Saharan countries are directly associated with the capacity of their related causative agents Toxoplasma and Plasmodium, respectively, to colonize and expand inside host cells. Therefore, deciphering how these two Apicomplexan protozoan parasites access their host cells has been highlighted as a priority research with the perspective of designing anti-invasive molecules to prevent diseases. Central to the mechanism of invasion for both genera is mechanical force, which is thought to be applied by the parasite at the interface between the two cells following assembly of a unique cell-cell junction but this model lacks direct evidence and has been challenged by recent genetic studies. In this work, using parasites expressing the fluorescent core component of this junction, we analyze characteristic features of the kinematics of penetration of more than 1,000 invasion events., Results: The majority of invasion events occur with a typical forward rotational progression of the parasite through a static junction into an invaginating host cell plasma membrane. However, if parasites encounter resistance and if the junction is not strongly anchored to the host cell cortex, as when parasites do not secrete the toxofilin protein and, therefore, are unable to locally remodel the cortical actin cytoskeleton, the junction travels retrogradely with the host cell membrane along the parasite surface allowing the formation of a functional vacuole. Kinetic measurements of the invasive trajectories strongly support a similar parasite driven force in both static and capped junctions, both of which lead to successful invasion. However, about 20% of toxofilin mutants fail to enter and eventually disengage from the host cell membrane while the secreted RhOptry Neck (RON2) molecules are posteriorally capped before being cleaved and released in the medium. By contrast in cells characterized by low cortex tension and high cortical actin dynamics junction capping and entry failure are drastically reduced., Conclusions: This kinematic analysis newly highlights that to invade cells parasites need to engage their motor with the junction molecular complex where force is efficiently applied only upon proper anchorage to the host cell membrane and cortex.

Objective: The protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability., Methods: The yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry., Results: We identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1., Conclusion: TgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth., (Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Apicomplexan parasites are thought to actively invade the host cell by gliding motility. This movement is powered by the parasite's own actomyosin system, and depends on the regulated polymerisation and depolymerisation of actin to generate the force for gliding and host cell penetration. Recent studies demonstrated that Toxoplasma gondii can invade the host cell in the absence of several core components of the invasion machinery, such as the motor protein myosin A (MyoA), the microneme proteins MIC2 and AMA1 and actin, indicating the presence of alternative invasion mechanisms. Here the roles of MyoA, MLC1, GAP45 and Act1, core components of the gliding machinery, are re-dissected in detail. Although important roles of these components for gliding motility and host cell invasion are verified, mutant parasites remain invasive and do not show a block of gliding motility, suggesting that other mechanisms must be in place to enable the parasite to move and invade the host cell. A novel, hypothetical model for parasite gliding motility and invasion is presented based on osmotic forces generated in the cytosol of the parasite that are converted into motility.

Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression., (© 2013 John Wiley & Sons Ltd.)

Cancer cells have an increased ability to squeeze through extracellular matrix gaps that they create by promoting proteolysis of its components. Major sites of degradation are specialized micro-domains in the plasma membrane collectively named invadosomes where the Arp2/3 complex and formin proteins cooperate to spatio-temporally control actin nucleation and the folding of a dynamic F-actin core. At invadosomes, proper coupling of exo-endocytosis allows polarized delivery of proteases that facilitate degradation of ECM and disruption of the cellular barrier. We investigated the contribution of the actin nucleator Spire-1 to invadosome structure and function, using Src-activated cells and cancer cells. We found that Spire-1 is specifically recruited at invadosomes and is part of a multi-molecular complex containing Src kinase, the formin mDia1 and actin. Spire-1 interacts with the Rab3A GTPase, a key player in the regulation of exocytosis that is present at invadosomes. Finally, over- and under-expression of Spire-1 resulted in cells with an increased or decreased potential for matrix degradation, respectively, therefore suggesting a functional interplay of Spire-1 with both actin nucleation and vesicular trafficking that might impact on cell invasive and metastatic behavior.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38α MAP kinase. This noncanonical kinetics of p38α activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24-p38α complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.

Apicomplexan parasites invade host cells by forming a ring-like junction with the cell surface and actively sliding through the junction inside an intracellular vacuole. Apical membrane antigen 1 is conserved in apicomplexans and a long-standing malaria vaccine candidate. It is considered to have multiple important roles during host cell penetration, primarily in structuring the junction by interacting with the rhoptry neck 2 protein and transducing the force generated by the parasite motor during internalization. Here, we generate Plasmodium sporozoites and merozoites and Toxoplasma tachyzoites lacking apical membrane antigen 1, and find that the latter two are impaired in host cell attachment but the three display normal host cell penetration through the junction. Therefore, apical membrane antigen 1, rather than an essential invasin, is a dispensable adhesin of apicomplexan zoites. These genetic data have implications on the use of apical membrane antigen 1 or the apical membrane antigen 1-rhoptry neck 2 interaction as targets of intervention strategies against malaria or other diseases caused by apicomplexans.

Toxoplasma gondii, a human pathogen and a model apicomplexan parasite, actively and rapidly invades host cells. To initiate invasion, the parasite induces the formation of a parasite-cell junction, and progressively propels itself through the junction, inside a newly formed vacuole that encloses the entering parasite. Little is known about how a parasite that is a few microns in diameter overcomes the host cell cortical actin barrier to achieve the remarkably rapid process of internalization (less than a few seconds). Using correlative light and electron microscopy in conjunction with electron tomography and three-dimensional image analysis we identified that toxofilin, an actin-binding protein, secreted by invading parasites correlates with localized sites of disassembly of the host cell actin meshwork. Moreover, quantitative fluorescence speckle microscopy of cells expressing toxofilin showed that toxofilin regulates actin filament disassembly and turnover. Furthermore, Toxoplasma tachyzoites lacking toxofilin, were found to be impaired in cortical actin disassembly and exhibited delayed invasion kinetics. We propose that toxofilin locally upregulates actin turnover thus increasing depolymerization events at the site of entry that in turn loosens the local host cell actin meshwork, facilitating parasite internalization and vacuole folding.

Apicomplexan zoites enter host cells by forming and actively moving through a tight junction (TJ) formed between the parasite and host cell surfaces. Although the TJ was first described decades ago, its molecular characterization has proved difficult mainly because of its transient existence during an internalization process that lasts only seconds. In the past 7 years, work has led to a model of the TJ in which the association between AMA1 and RON proteins structures the TJ and bridges the cytoskeletons of the two cells. However, more recent work questions this view. Here, we critically discuss the current model and speculate on alternative models of the AMA1-RON association and of the apicomplexan TJ., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Streptococcus agalactiae (group B Streptococcus, GBS), a normal constituent of the intestinal microbiota is the major cause of human neonatal infections and a worldwide spread 'hypervirulent' clone, GBS ST-17, is strongly associated with neonatal meningitis. Adhesion to epithelial and endothelial cells constitutes a key step of the infectious process. Therefore GBS surface-anchored proteins are obvious potential adhesion mediators of barrier crossing and determinant of hypervirulence. This review addresses the most recent molecular insights gained from studies on GBS surface proteins proven to be involved in the crossing of the brain-blood barrier and emphasizes on the specificity of a hypervirulent clone that displays meningeal tropism., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Streptococcus agalactiae (group B streptococcus; GBS) is a normal constituent of the intestinal microflora and the major cause of human neonatal meningitis. A single clone, GBS ST-17, is strongly associated with a deadly form of the infection called late-onset disease (LOD), which is characterized by meningitis in infants after the first week of life. The pathophysiology of LOD remains poorly understood, but our epidemiological and histopathological results point to an oral route of infection. Here, we identify a novel ST-17-specific surface-anchored protein that we call hypervirulent GBS adhesin (HvgA), and demonstrate that its expression is required for GBS hypervirulence. GBS strains that express HvgA adhered more efficiently to intestinal epithelial cells, choroid plexus epithelial cells, and microvascular endothelial cells that constitute the blood-brain barrier (BBB), than did strains that do not express HvgA. Heterologous expression of HvgA in nonadhesive bacteria conferred the ability to adhere to intestinal barrier and BBB-constituting cells. In orally inoculated mice, HvgA was required for intestinal colonization and translocation across the intestinal barrier and the BBB, leading to meningitis. In conclusion, HvgA is a critical virulence trait of GBS in the neonatal context and stands as a promising target for the development of novel diagnostic and antibacterial strategies.

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.

Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.

The invasive stages of Apicomplexa parasites, called zoites, have been largely studied in in vitro systems, with a special emphasis on their unique gliding and host cell invasive capacities. In contrast, the means by which these parasites reach their destination in their hosts are still poorly understood. We summarize here our current understanding of the cellular basis of in vivo parasitism by two well-studied Apicomplexa zoites, the Toxoplasma tachyzoite and the Plasmodium sporozoite. Despite being close relatives, these two zoites use different strategies to reach their goal and establish infection.

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion. Toxofilin is found in rhoptry and presents an N-terminal signal sequence, consistent with its being secreted during invasion. We report the structure of toxofilin amino acids 69-196 in complex with the host mammalian actin. Toxofilin presents an extended conformation and interacts with an antiparallel actin dimer, in which one of the actins is related by crystal symmetry. Consistent with this observation, analytical ultracentrifugation analysis shows that toxofilin binds two actins in solution. Toxofilin folds into five consecutive helices, which form three relatively independent actin-binding sites. Helices 1 and 2 bind the symmetry-related actin molecule and cover its nucleotide-binding cleft. Helices 3-5 bind the other actin and constitute the primary actin-binding region. Helix 3 interacts in the cleft between subdomains 1 and 3, a common binding site for most ABPs. Helices 4 and 5 wrap around actin subdomain 4, and residue Gln-134 of helix 4 makes a hydrogen-bonding contact with the nucleotide in actin, both of which are unique features among ABPs. Toxofilin dramatically inhibits nucleotide exchange on two actin molecules simultaneously. This effect is linked to the formation of the antiparallel actin dimer because a construct lacking helices 1 and 2 binds only one actin and inhibits nucleotide exchange less potently.