Fostering innovation to solve the biomechanics of microbe-host interactions: Focus on the adhesive forces underlying Apicomplexa parasite biology.

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.

Identification of new components of the basal pole of Toxoplasma gondii provides novel insights into its molecular organization and functions.

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.

The BCC7 Protein Contributes to the Toxoplasma Basal Pole by Interfacing between the MyoC Motor and the IMC Membrane Network.

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 Leishmania donovani LDBPK_220120.1 Gene Encodes for an Atypical Dual Specificity Lipid-Like Phosphatase Expressed in Promastigotes and Amastigotes; Substrate Specificity, Intracellular Localizations, and Putative Role(s).

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 LdTyrPIP_22 (Leishmaniad onovani Tyrosine PI Phosphatase, gene locus at chromosome 22). By immunofluorescence confocal microscopy we localized the LdTyrPIP_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 LdTyrPIP_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.

A brain cyst load-associated antigen is a Toxoplasma gondii biomarker for serodetection of persistent parasites and chronic infection.

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.

Parasitism as a lifestyle: Ultimate intimacy between Apicomplexan protozoans and metazoan hosts.

Editorial: The Apicomplexa parasite Toxoplasma gondii glides on substrate with a helical path and releases material that forms a trail behind. The helical microtubules (green) periodically compress and relax, acting as spring force by coupling with the myosin motor (red).

Profiling of myristoylation in Toxoplasma gondii reveals an N-myristoylated protein important for host cell penetration.

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.

A microscopic parasite known as Toxoplasma gondii infects around 30% of the human population. Most infections remain asymptomatic, but in people with a compromised immune system, developing fetuses and people infected with particular virulent strains of the parasite, infection can be fatal. T. gondii is closely related to other parasites that also infect humans, including the one that causes malaria. These parasites have complex lifecycles that involve successive rounds of invading the cells of their hosts, growing and then exiting these cells. Signaling proteins found at specific locations within parasite cells regulate the ability of the parasites to interact with and invade host cells. Sometimes these signaling proteins are attached to membranes using lipid anchors, for example through a molecule called myristic acid. An enzyme called NMT can attach myristic acid to one end of its target proteins. The myristic acid tag can influence the ability of target proteins to bind to other proteins, or to membranes. Previous studies have found that drugs that inhibit the NMT enzyme prevent the malaria parasite from successfully invading and growing inside host cells. The NMT enzyme from T. gondii is very similar to that of the malaria parasite. Broncel et al. have shown that the drug developed against P. falciparum also inhibits the ability of T. gondii to grow. These findings suggest that drugs against the NMT enzyme may be useful to treat diseases caused by T. gondii and other closely-related parasites. Broncel et al. also identified 65 proteins in T. gondii that contain a myristic acid tag using an approach called proteomics. One of the unexpected 'myristoylated' proteins identified in the experiments is known as MIC7. This protein was found to be transported onto the surface of T. gondii parasites and is required in its myristoylated form for the parasite to successfully invade host cells. This was surprising as myristoylated proteins are generally thought to not enter the pathway that brings proteins to the outside of cell. These findings suggest that myristic acid on proteins that are secreted can facilitate interactions between cells, maybe by inserting the myristic acid into the cell membrane.

Coupling Polar Adhesion with Traction, Spring, and Torque Forces Allows High-Speed Helical Migration of the Protozoan Parasite Toxoplasma.

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.

Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication.

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.

Francisella novicida and F. philomiragia biofilm features conditionning fitness in spring water and in presence of antibiotics.

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.

Phenotyping Toxoplasma Invasive Skills by Fast Live Cell Imaging.

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.

Apicomplexan F-actin is required for efficient nuclear entry during 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.

Intracellular protozoan parasites: living probes of the host cell surface molecular repertoire.

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.

The Toxoplasma effector TEEGR promotes parasite persistence by modulating NF-κB signalling via EZH2.

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.

Specific Targeting of Plant and Apicomplexa Parasite Tubulin through Differential Screening Using In Silico and Assay-Based Approaches.

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.

Characterization of a Toxoplasma effector uncovers an alternative GSK3/ß-catenin-regulatory pathway of inflammation.

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 ß-catenin destruction complex. By interacting with GSK3/PP2A-B56, GRA18 drives ß-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/ß-catenin axis to selectively reprogram immune gene expression.

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.

Modifications at K31 on the lateral surface of histone H4 contribute to genome structure and expression in apicomplexan parasites.

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.

Actin Nanobodies Uncover the Mystery of Actin Filament Dynamics in Toxoplasma gondii.

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.