Spire-1 contributes to the invadosome and its associated invasive properties.

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.

AMA1-deficient Toxoplasma gondii parasites transiently colonize mice and trigger an innate immune response that leads to long-lasting protective immunity.

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.

The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force.

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.

Toxofilin upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion.

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.

Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites.

Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full-length TgSUB1 restores processing, complementation of Δsub1 parasites with TgSUB1 lacking the GPI anchor (Δsub1::ΔGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Δsub1 and Δsub1::ΔGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.

The prodomain of Toxoplasma gondii GPI-anchored subtilase TgSUB1 mediates its targeting to micronemes.

Subtilisin-like proteases have been proposed to play an important role for parasite survival in Toxoplasma gondii (Tg) and Plasmodium falciparum. The T. gondii subtilase TgSUB1 is located in the microneme, an apical secretory organelle whose contents mediate adhesion to the host during invasion. TgSUB1 is predicted to contain a glycosyl-phosphatidylinositol (GPI) anchor. This is unusual as Toxoplasma GPI-anchored proteins are targeted to the parasite's surface. In this study, we report that the subtilase TgSUB1 is indeed a GPI-anchored protein but contains dominant microneme targeting signals. Accurate targeting of TgSUB1 to the micronemes is dependent upon several factors including promoter strength and timing, accurate processing and folding. We analyzed the targeting domains of TgSUB1 using TgSUB1 deletion constructs and chimeras made between TgSUB1 and reporter proteins. The TgSUB1 prodomain is responsible for trafficking to the micronemes and is sufficient for targeting a reporter protein to the micronemes. Trafficking is dependent upon correct folding or other context-dependent conformation as the prodomain expressed alone is unable to reach the micromenes. Therefore, TgSUB1 is a novel example of a GPI-anchored protein in T. gondii that bypasses the GPI-dependent surface trafficking pathway to traffic to micronemes, specialized regulated secretory organelles.

Toxoplasma Parasite Twisting Motion Mechanically Induces Host Cell Membrane Fission to Complete Invasion within a Protective Vacuole.

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.

Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity.

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is transmitted through tick bite. Lyme borreliosis evolves in two stages: a primary red skin lesion called erythema migrans; later on, invasive bacteria disseminate to distant sites inducing secondary manifestations (neuropathies, arthritis, carditis, late skin disorders). It has been previously suggested that the ospC gene could be associated with invasiveness in humans depending on its sequence. Here, we confirm the pattern of invasiveness, according to B. burgdorferi sensu stricto (B. b. ss) ospC group, using the mouse as an experimental host of B. b. ss. As it has been shown that the host plasminogen activation system is used by B. burgdorferi to disseminate throughout the host, we studied the interaction of plasminogen with OspC proteins from invasive and non-invasive groups of B. b. ss. Using two methods, ELISA and surface plasmon resonance, we demonstrate that indeed OspC is a plasminogen-binding protein. Moreover, significant differences in binding affinity for plasminogen are correlated with different invasiveness patterns in mice. These results suggest that the correlation between ospC polymorphism and Borrelia invasiveness in humans is linked, at least in part, to differences in OspC affinity for plasminogen.

Molecular diversity of the ospC gene in Borrelia. Impact on phylogeny, epidemiology and pathology.

Lyme borreliosis is a 2 steps disease: i) Localized erythema migrans ii) occasionally a disseminated disease. Three out of the 10 up to now described Borrelia species are pathogenic for man and each of them exhibits its own organotropism: joints for Borrelia burgdorferi sensu stricto (B.b. ss), nervous system for B. garinii, skin for B. afzelii, ospc gene is subject to lateral transfer leading to a huge diversity among corresponding encoded proteins. This allows the spirochete to develop a repertoire of epitopes to escape the host immune response. We noticed that the European endemic ospc repertoire is only a subset of the American one. This bottleneck situation transduces a "founder's event" suggesting B.b. ss has been imported from North America to Europe at historical times. Another valuable observation is the fact that isolates from disseminated forms (called "invasive") of the disease, all are distributed in only ten out of the 70 ospc genotypes. The conclusion is that in human, some OspC conformations are associated with the invasive potential of a given Borrelia isolate.

Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion.

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.

Host cell invasion by apicomplexans: what do we know?

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.

Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa.

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.

Genetic diversity among Borrelia strains determined by single-strand conformation polymorphism analysis of the ospC gene and its association with invasiveness.

Lyme borreliosis (LB) is a tick-borne spirochetal infection caused by three Borrelia species: Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto. LB evolves in two stages: a skin lesion called erythema migrans and later, different disseminated forms (articular, neurological, cardiac.). Previous research based on analysis of ospC sequences allowed the definition of 58 groups (divergence of <2% within a group and >8% between groups). Only 10 of these groups include all of the strains isolated from disseminated forms that are considered invasive. The aim of this study was to determine whether or not invasive strains belong to restricted ospC groups by testing human clinical strains isolated from disseminated forms. To screen for ospC genetic diversity, we used single-strand conformation polymorphism (SSCP) analysis. Previously known ospC sequences from 44 different strains were first tested, revealing that each ospC group had a characteristic SSCP pattern. Therefore, we studied 80 disseminated-form isolates whose ospC sequences were unknown. Of these, 28 (35%) belonged to previously known invasive groups. Moreover, new invasive groups were identified: six of B. afzelii, seven of B. garinii, and one of B. burgdorferi sensu stricto. This study confirmed that invasive strains are not distributed among all 69 ospC groups but belong to only 24 groups. This suggests that OspC may be involved in the invasiveness of B. burgdorferi.