Mycobacterium abscessus is a rapidly growing Mycobacterium causing a wide spectrum of clinical syndromes. It now is recognized as a pulmonary pathogen to which cystic fibrosis patients have a particular susceptibility. The M. abscessus rough (R) variant, devoid of cell-surface glycopeptidolipids (GPLs), causes more severe clinical disease than the smooth (S) variant, but the underlying mechanisms of R-variant virulence remain obscure. Exploiting the optical transparency of zebrafish embryos, we observed that the increased virulence of the M. abscessus R variant compared with the S variant correlated with the loss of GPL production. The virulence of the R variant involved the massive production of serpentine cords, absent during S-variant infection, and the cords initiated abscess formation leading to rapid larval death. Cording occurred within the vasculature and was highly pronounced in the central nervous system (CNS). It appears that M. abscessus is transported to the CNS within macrophages. The release of M. abscessus from apoptotic macrophages initiated the formation of cords that grew too large to be phagocytized by macrophages or neutrophils. This study is a description of the crucial role of cording in the in vivo physiopathology of M. abscessus infection and emphasizes cording as a mechanism of immune evasion.morphotype | pathogenesis | granuloma | innate immunity T he rapidly growing mycobacterium (RGM) Mycobacterium abscessus (M. abscessus) is an emerging pathogen that infects a wide spectrum of tissues in humans, including lungs, skin, and soft tissues (1, 2). M. abscessus lung disease is highly prevalent in patients with cystic fibrosis (CF) and is becoming a major issue for most CF centers worldwide (3-6). Although M. abscessus is an RGM, it can persist and cause lung disease with caseous lesions (7).M. abscessus exists as two variants: rough (R) and smooth (S). Ex vivo and in vivo studies have described the hypervirulence phenotype of the R versus the S morphotype (8, 9), and epidemiological studies have confirmed the persistence and acute respiratory syndromes caused by the R morphotype (4, 10, 11). The major difference between the R and S variants is the loss of a surface-associated glycopeptidolipid (GPL) (12). Analysis of the pathogenicity of M. abscessus has been hampered by the lack of genetic tools and the restricted panel of cellular/animal models. However, new genetic tools, including conditional gene expression, recently have been applied to both the S and R morphotypes (13, 14), but developing new animal models amenable to the manipulation of the host response is still challenging. The M. abscessus genome harbors a mercury-resistance plasmid sharing 99% identity with an episome from the slowgrowing fish pathogen Mycobacterium marinum, indicating that these species have exchanged this plasmid in a shared ecosystem (15). M. abscessus has been described in wild and captive fish species (16,17), and hand infections caused by M. abscessus have been reported in healthy fish handlers (18), sug...

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

| Protozoan parasites have developed elaborate motility systems that facilitate infection and dissemination. For example, amoebae use actin-rich membrane extensions called pseudopodia, whereas Kinetoplastida are propelled by microtubule-containing flagella. By contrast, the motile and invasive stages of the Apicomplexa -a phylum that contains the important human pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis) -have a unique machinery called the glideosome, which is composed of an actomyosin system that underlies the plasma membrane. The glideosome promotes substrate-dependent gliding motility, which powers migration across biological barriers, as well as active host cell entry and egress from infected cells. In this Review, we discuss the discovery of the principles that govern gliding motility, the characterization of the molecular machinery involved, and its impact on parasite invasion and egress from infected cells. NATURE REVIEWS | MICROBIOLOGYADVANCE ONLINE PUBLICATION | 1 REVIEWS © 2 0 1 7 M a c m i l l a n P u b l i s h e r s L i m i t e d , p a r t o f S p r i n g e r N a t u r e . A l l r i g h t s r e s e r v e d . ToxoplasmosisA food-borne infection caused by the parasite Toxoplasma gondii. The infection is usually mild or even asymptomatic, but can have serious consequences in patients who are immunocompromised and for the fetus in the case of primary infection during pregnancy. SporozoitesThe infectious and motile stage produced in oocysts and transmitted by the definitive host. TachyzoitesThe motile and fast-replicative stage of Toxoplasma gondii that is able to invade any nucleated cell in the host. MerozoitesThe stage of Plasmodium spp. that infects erythrocytes, in which it initiates a new asexual life cycle. Actomyosin systemA complex that comprises actin filaments, myosin and associated proteins, and that is involved in movement. GlideosomeA molecular complex that powers gliding motility in apicomplexan parasites.motility, invasion and egress. In this Review, we focus primarily on the T. gondii tachyzoite, for which functional studies were first carried out, and we compare and contrast this with the motile stages of malaria parasites -the sporozoite, merozoite and ookinete.Emergence of the capping model The motility of Apicomplexa (historically named Sporozoa) has caught the attention of scientists for more than a century 8 and was named gliding motility early on, on the basis of microscopic observations Nature Reviews | Microbiology www.nature.com/nrmicro © 2 0 1 7 M a c m i l l a n P u b l i s h e r s L i m i t e d , p a r t o f S p r i n g e r N a t u r e . A l l r i g h t s r e s e r v e d . of live specimens (FIG. 2; Supplementary information S1-S5 (movies)). This mode of motility differs substantially to amoeboid motion involving pseudopods, and from the ciliary and flagellar motion used by most unicellular organisms. The main hypothesis to explain gliding motility in the early days was slime secretion, as seen in slugs; ...

Toxoplasma gondii is a single-celled, eukaryotic parasite that can only reproduce inside a host cell. Upon entry, this Apicomplexan parasite co-opts host functions for its own purposes. An unusual set of apical organelles, named rhoptries, contain some of the machinery that is used by T. gondii both for invasion and to commandeer host functions. Of particular interest are a group of injected protein kinases that are among the most variable of all the T. gondii proteins. At least one of these kinases has a major effect on host-gene expression, including the modulation of key regulators of the immune response. Here, we discuss these recent findings and use them to propose a model in which an expansion of host range is a major force that drives rhoptry-protein evolution.

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.

The malaria sporozoite, the parasite stage transmitted by the mosquito, is delivered into the dermis and differentiates in the liver. Motile sporozoites can invade host cells by disrupting their plasma membrane and migrating through them (termed cell traversal), or by forming a parasite-cell junction and settling inside an intracellular vacuole (termed cell infection). Traversal of liver cells, observed for sporozoites in vivo, is thought to activate the sporozoite for infection of a final hepatocyte. Here, using Plasmodium berghei, we show that cell traversal is important in the host dermis for preventing sporozoite destruction by phagocytes and arrest by nonphagocytic cells. We also show that cell infection is a pathway that is masked, rather than activated, by cell traversal. We propose that the cell traversal activity of the sporozoite must be turned on for progression to the liver parenchyma, where it must be switched off for infection of a final hepatocyte.

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

SummaryMost Apicomplexa are obligate intracellular parasites and many are important pathogens of human and domestic animals. For a successful cell invasion, they rely on their own motility and on a firm anchorage to their host cell, depending on the secretion of proteins and the establishment of a structure called the moving junction (MJ). The MJ moves from the apical to the posterior end of the parasite, leading to the internalization of the parasite into a parasitophorous vacuole. Based on recent data obtained in Plasmodium and Toxoplasma, an emerging model emphasizes a cooperative role of secreted parasitic proteins in building the MJ and driving this crucial invasive process. More precisely, the parasite exports the microneme protein AMA1 to its own surface and the rhoptry neck RON2 protein as a receptor inserted into the host cell together with other RON partners. Ongoing and future research will certainly help refining the model by characterizing the molecular organization within the MJ and its interactions with both host and parasite cytoskeleton for anchoring of the complex.