© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs232488. doi:10.1242/jcs.232488

REVIEW SUBJECT COLLECTION: CELL BIOLOGY AND DISEASE Cell invasion by intracellular parasites – the many roads to infection Maria Fátima Horta1, Luciana Oliveira Andrade2, Érica Santos Martins-Duarte3 and Thiago Castro-Gomes3,*

ABSTRACT physiological host cell processes, such as lysosome-triggered 2+ Intracellular parasites from the genera Toxoplasma, Plasmodium, Ca -dependent endocytosis, which is used by all nucleated cells Trypanosoma, Leishmania and from the phylum Microsporidia are, to repair wounded PMs (Corrotte and Castro-Gomes, 2019); this respectively, the causative agents of toxoplasmosis, malaria, Chagas pathway has previously shown to occur for Trypanosoma cruzi disease, leishmaniasis and microsporidiosis, illnesses that kill millions (Rodríguez et al., 1996; Fernandes et al., 2011), and also more of people around the globe. Crossing the host cell plasma membrane recently by our lab for Leishmania amazonensis (Cavalcante-Costa (PM) is an obstacle these parasites must overcome to establish et al., 2019). Large parasites can be equally phagocytosed, if they themselves intracellularly and so cause diseases. The mechanisms of are able to resist phagocytic degradation and live within phagocytic cell invasion are quite diverse and include (1) formation of moving cells, such as protozoans of the genus Leishmania (Zenian et al., junctions that drive parasites into host cells, as for the protozoans 1979). Finally, internalization can also be induced through the Toxoplasma gondii and Plasmodium spp., (2) subversion of endocytic formation of a specialized structure that directs the parasite towards pathways used by the host cell to repair PM, as for Trypanosoma cruzi the PM, as for microsporidians, for example, Encephalitozoon spp. and Leishmania, (3) induction of phagocytosis as for Leishmania or (Xu and Weiss, 2005). Understanding the initial interaction between (4) endocytosis of parasites induced by specialized structures, such as a host cell and an intracellular parasite is essential, as the molecular the polar tubes present in microsporidian species. Understanding the determinants involved could be exploited as vaccine targets to help early steps of cell entry is essential for the development of vaccines block the infection from the onset. This Review will focus on the and drugs for the prevention or treatment of these diseases, and thus cellular and molecular determinants that mediate host cell invasion enormous research efforts have been made to unveil their underlying by the above parasites paying special attention to the initial steps. biological mechanisms. This Review will focus on these mechanisms – and the factors involved, with an emphasis on the recent insights into Apicomplexans Plasmodium spp. and Toxoplasma gondii the cell biology of invasion by these pathogens. Apicomplexans are obligate intracellular parasites characterized by the presence of an apical complex, a cellular structure crucial for cell KEY WORDS: Host cell invasion, Plasmodium spp., Toxoplasma invasion composed of cytoskeletal components and three secretory gondii, Trypanosoma cruzi, Microsporidia organelles, namely, micronemes, rhoptries and dense granules (Adl et al., 2018). They also lack flagella, cilia or pseudopods, thus Introduction relying on a specialized intracellular machinery that allows them to Evolving a way to invade their host cells is the first obligatory move when attached to substrates or the host cell PM. Toxoplasma challenge for intracellular parasites. In order to establish gondii (agent of toxoplasmosis) and Plasmodium spp. (agents of intracellular life, these microorganisms employ numerous malaria) are included within this phylum (Fig. 1; Box 1). strategies to overcome the barrier that is imposed by the host cell plasma membrane (PM) and cytoskeleton. Small intracellular Cell invasion by apicomplexans pathogens, such as bacteria and viruses, can be endocytosed Owing to their inability to undergo cell division and limited viability through natural and abundant PM invaginations, via clathrin-, in extracellular environments, apicomplexans have evolved a unique caveolae- and flotillin-mediated endocytosis (Cossart and Helenius, and extremely effective mechanism for invading a host cell, which is 2014). However, these small, nanoscale membrane invaginations dependent on gliding motility and coordinated secretion of proteins cannot carry larger intracellular parasites, such as protozoans. The contained in their apical secretory organelles (Box 2) (Carruthers and internalization of these pathogens can be achieved instead through Sibley, 1997). In the initial moments of interaction with the host cell, several other routes. Larger pathogens can actively drive cell entry the apicomplexan zoite (see Glossary) moves laterally on its surface through the formation of a specialized cell machinery, such as the until it encounters an ideal receptor that triggers host cell invasion; moving junction that is present in the apicomplexans Plasmodium this then leads to an apical re-orientation of the zoite, which is spp. (Aikawa et al., 1978) and Toxoplasma gondii (Mordue et al., mediated by microneme adhesins and is followed by the secretion of 1999). Invasion can also occur through the subversion of rhoptry proteins into host cell cytoplasm through a transient pore in the host cell PM (Carruthers and Boothroyd, 2007; Weiss et al., 2015;

1Departamento de Bioquımicá e Imunologia, Instituto de Ciências Biológicas, Dubremetz, 2007) (see Fig. 4A). Universidade Federal de Minas Gerais, Belo Horizonte, CEP 31270-901, Brazil. In T. gondii, the initial lateral weak interaction with host 2Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade cell membrane is mediated by glycosylphosphatidylinositol (GPI)- Federal de Minas Gerais, Belo Horizonte, CEP 31270-901, Brazil. 3Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas anchored proteins of the surface antigen glycoprotein (SAG)-related Gerais, Belo Horizonte, CEP 31270-901, Brazil. sequence (SRS) superfamily, which are resident on the parasite PM (Dzierszinski et al., 2000; Manger et al., 1998; Wasmuth et al., 2012). *Author for correspondence ([email protected]) SRS genes are differently expressed according to lineage or

T.C., 0000-0003-1564-4645 evolutionary stage, and are involved in a wide range of functions, Journal of Cell Science

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A

B Dense granules 1 Micronemes

2

Toxoplasma gondii

Rhoptries

Host cell PM Extracellular Intracellular

Rhoptry- Moving secreted junction Host cell proteins proteins

Parasite PM IM C Host cell PM Key

Host cell proteins Parasite proteins

Actin Actin cytoskeleton motor RON5 Parasite direct ALIX AMA1 RON8

ion Motor d ir RON2 GAC ection CIN85/CD2AP ROM 4 TSG101 RON4 Moving junction

Fig. 1. Apicomplexans invade cells by forming a tight moving junction with host cell PMs. (A) Erythrocyte invasion by a P. knowlesi merozoite. Left, an electron micrograph of a merozoite of P. knowlesi at the initial contact between the merozoite’s apical end (arrow) and an erythrocyte (E). The merozoite shows an apical end (labeled A), a rhoptry (R), a nucleus (N) and a mitochondrion (M). The surface is covered with a surface coat (double arrow). Middle, the erythrocyte membrane is thickened (15 nm) at the attachment site (arrow). Inset, higher magnification micrograph of the erythrocyte–merozoite attachment site showing the thickened erythrocyte membrane. Right, an advanced stage of erythrocyte (E) entry by a merozoite (Mz). Note a junctional attachment (labeled C) at each side of the entry orifice. Republished with permission of Rockefeller University Press from Aikawa et al., 1978; permission conveyed through the Copyright Clearance Center. (B) Schematic representation of cell binding and formation of the moving junction during host cell invasion by the apicomplexan parasite Toxoplasma gondii. (1) Rhoptry secretion. After re-orientation, the parasite apical complex faces the host cell PM. A local loosening of the host cell actin cytoskeleton is induced by the injection of parasite profilin, and rhoptry proteins are discharged into host cell cytoplasm. (2) Moving junction formation (see also enlargement and electron micrograph underneath). Proteins secreted by the parasite and host cell proteins form a multi-molecular complex beneath host cell PM. This is seen in the electron micrograph (É.S.M.-D.) as an electron-dense region (black arrow) in a host cell (HC; here, a LLC-MK2 epithelial cell) being invaded by a T. gondii (Tg) tachyzoite. This is the region where the moving junction is formed. The enlargement shows a schematic representation of the moving junction. Rhoptry proteins (RON2, RON4, RON5 and RON8) form a molecular complex with host cell proteins of the ESCRT family (ALIX, TSG101, CIN85 and CD2AP); this anchors the parasite to the host cell actin cytoskeleton, while the extracellular domain of RON2 binds to AMA1 present on parasite PM. Inside the parasite, the cytosolic domain of AMA1 binds to a parasite actin-myosin motor through the glideosome-associated connector (GAC), while the rhomboid protease ROM4 successively disengages the complex to allow parasite moving. IMC, inner membrane complex. See also Box 2. Journal of Cell Science

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Box 1. Transmission and parasite biology of T. gondii, Box 2. Apicomplexans – microneme and rhoptry secretion Plasmodium spp., T. cruzi, Leishmania spp. and and gliding motility during host cell invasion microsporidians Microneme protein secretion in T. gondii and Plasmodium involves Ca2+ T. gondii is transmitted through ingestion of sporozoite-containing signaling (Dawn et al., 2014; Wetzel et al., 2004) that is initiated by its oocysts from felids or encysted bradyzoites present in infected mobilization from intracellular stores by cGMP (T. gondii) (Bullen et al., undercooked meat. After ingestion, sporozoites or bradyzoites infect 2016) or cAMP (Plasmodium) (Dawn et al., 2014) signaling. Ca2+ gastrointestinal epithelial cells in the ileum (Dubey, 2007; Dubey et al., increase in the cytosol activates Ca2+-dependent protein kinases, 1997; Speer and Dubey, 1998). Bradyzoites or sporozoites then regulating microneme secretion (Lourido et al., 2010; Wetzel et al., differentiate into tachyzoites, which spread throughout the body, 2004; Singh et al., 2010; Wernimont et al., 2010) as well as microneme generating pathology (Dubey et al., 1997; Speer and Dubey, 1998). and PM fusion, which is mediated by the parasite protein DOC2.1 protein Infection by Plasmodium spp. occurs through the inoculation of (Farrell et al., 2012). In T. gondii, phosphatidic acid also participates in sporozoites into the skin of vertebrate hosts by anopheline mosquitoes this secretion process by binding to the acylated pleckstrin homology (Matsuoka et al., 2002; Sidjanski and Vanderberg, 1997). Sporozoites domain-containing protein (APH) that is present in micronemes (Bullen then migrate to the liver and infect hepatocytes, in which they replicate et al., 2016). Rhoptry secretion is followed by strong binding of the (reviewed by Yang and Boddey, 2017), originating merozoites. After parasite to the host cell surface, which is mediated by specific reaching the bloodstream, merozoites invade erythrocytes, in which they microneme-secreted adhesins (Kessler et al., 2008; Singh et al., are able to replicate and, thus, generate additional merozoites that can 2010). In T. gondii, MIC8 appears to participate in rhoptry secretion be released and cyclically invade new erythrocytes, thereby giving rise to (Kessler et al., 2008), while in Plasmodium, the interaction of the the erythocytic cycle of malaria. microneme adhesin EBA175 with its receptor glycophorin A decreases T. cruzi is transmitted to humans as flagellated metacyclic cytosolic Ca2+ levels in merozoites and triggers rhoptry secretion (Singh trypomastigotes by kissing-bug insects of the Reduviidae family et al., 2010). A cytoplasmic protein homologous to the ferlin family of (Coura and Dias, 2009; Araújo et al., 2009). These forms evolve in the Ca2+-sensing proteins (TgFER2) has been shown to be essential in invertebrate gut from replicative forms, the epimastigotes. After blood rhoptry secretion in T. gondii and points to a role for Ca2+ signaling in the repast, the vector feces containing trypomastigotes come into contact secretion of this organelle during invasion (Coleman et al., 2018). In with either the mucosa or skin micro-lesions at the bite site. addition, a newly identified set of proteins termed rhoptry apical surface Trypomastigotes can then invade any nucleated cell, where they proteins (RASPs) appear to be essential for rhoptry secretion in both transform into replicative oval-shaped forms with unapparent flagella, T. gondii and P. falciparum by promoting the rhoptry fusion with parasite the amastigotes, which differentiate again into trypomastigotes. These PM in a Ca2+-independent manner (Suarez et al., 2019). forms can invade neighboring cells or reach the bloodstream, thereby Gliding is a substrate-dependent motility that is mediated by a protein either spreading to other tissues or being ingested by a new insect vector complex, called the glideosome (Opitz and Soldati, 2002), which has an (Brener, 1973). actomyosin motor that is composed of myosin A, myosin light chain, Leishmania spp. are transmitted by the bite of a sand fly vector, which myosin essential chain and actin filaments (Boucher and Bosch, 2015). inoculates flagellated infective parasites, the metacyclic promastigotes, Motility is generated after the displacement of the myosin A head from the into the dermis of vertebrate hosts (Handman, 1999). Promastigotes can actin filament, which occurs when the extracellular domain of a transiently invade different cell types (Peters et al., 2008; Williams, 1988; transmembrane micronemal protein (TRAP in Plasmodium and MIC2 Kautz-Neu et al., 2012; Moll et al., 1993; Bogdan et al., 2000; in T. gondii) strongly interacts with a receptor at the host cell surface or in Cavalcante-Costa et al., 2019), before reaching macrophages, their the extracellular matrix. A physical interaction between the cytoplasmic final destination. Inside macrophages, they nest within vacuoles, where tail of TRAP or MIC2 and the actin filament is mediated by a conserved they transform into replicative oval-shaped amastigote forms, causing protein, named glideosome-associated connector (GAC) (Jacot et al., different clinical manifestations of the disease, depending on the species 2016). Thus, gliding is the result of the displacement of myosin A from of Leishmania and on host immune responses. actin filaments, followed by the release of the parasite from the host cell, Infection by microsporidians is established through the inhalation or after the proteolytic removal of TRAP or MIC2 by an intramembrane ingestion of virulent spores, which can infect several cell types (reviewed serine protease of the rhomboid family protease (ROM4) (reviewed by by Han and Weiss, 2017). Upon contact with host cells, the spores extrude Frénal et al., 2017), which then propels the parasite body forward. a long infection apparatus, the polar tube, which drives the parasite into the host cell. Parasites are internalized in a PV, where it replicates, generating more spores, which leave the host cell to infect neighboring cells, thereby amplifying infection (CDC - DPDx - Microsporidiosis; https://www.cdc.gov/ and merozoite (see Glossary) surface proteins (MSPs) (reviewed by dpdx/microsporidiosis/index.html). Beeson et al., 2016). CSP is highly conserved among Plasmodium species and mediates sporozoite (see Glossary) migration from mammalian dermis to liver (Tewari et al., 2002). Upon reaching the liver, sporozoites switch from a migratory to an invasive mode. including cell adhesion (Dzierszinski et al., 2000; Kim et al., 2007; Sporozoite binding to heparan sulfate proteoglycans (HSPGs) that Leal-Sena et al., 2018; Tomita et al., 2018; Wasmuth et al., 2012). are expressed by hepatic stellate cells culminates in the secretion of Interaction of SRS proteins with the host cell or substrates is, cysteine proteases by the parasite (Coppi et al., 2005; Frevert et al., however, weak, possibly allowing the parasite to move laterally along 1993; Pinzon-Ortiz et al., 2001; Zhao et al., 2016). This leads to a their surface (Carruthers and Boothroyd, 2007). Disruption of the proteolytic processing of CSP, exposing its thrombospondin type-1 SAG3 gene, which codes for the SRS adhesive protein, reduces repeat (TSR) domain, leading to an increase in parasite adhesion, parasite adhesion to host cell and also decreases virulence of T. gondii which allows invasion (Coppi et al., 2005). The weak interaction of to mice (Dzierszinski et al., 2000). It has also been shown that both merozoites with erythrocytes is mediated by MSPs (Gilson et al., SAG3 and SAG2 interact with heparin sulfate proteoglycans (Jacquet 2006). MSP1 is the most abundant and well-studied MSP (Cowman et al., 2001; Zhang et al., 2019). SAG1 also interacts with sulfated and Crabb, 2006), and is considered an important target for vaccine proteoglycans, as supported by structural studies and cell binding development (Beeson et al., 2016). MSP1 is proteolytically assays (He et al., 2002; Azzouz et al., 2013). processed by the subtilisin-like protease 1 into four fragments The initial interaction between Plasmodium zoites and host cells (denoted p42, p38, p30 and p83) (Koussis et al., 2009), which have also rely on parasite GPI-anchored proteins (Beeson et al., 2016), been implicated in the binding to the erythrocyte surface proteins such as the circumsporozoite protein (CSP) (Yoshida et al., 1980) band 3 (also known as SLC4A1) and glycophorin A (Baldwin et al., Journal of Cell Science

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for hepatocyte invasion (Ishino et al., 2005). P52 is a membrane Glossary GPI-anchored protein and acts as scaffold for P36 (Arredondo et al., Zoite: a general name for the infective forms of Apicomplexan parasites 2018). This scaffold is then responsible for the interaction with that invade cells. different receptors at the hepatocyte surface, such as CD81 and Oocyst: infective form of T. gondii delivered in feline feces (which scavenger receptor BI (SR-BI, also known as SCARB1) for contains infective sporozoites) that can be ingested by the mammalian P. falciparum and P. vivax sporozoites, respectively (Manzoni host. Bradyzoite: infective and resistant form of T. gondii found in host-tissue et al., 2017). Regarding merozoites, here, proteins belonging to the cysts that can be ingested by the mammalian host. Duffy binding-like family (DBL), reticulocyte binding-like (RBL) Tachyzoite: infective and replicative form of T. gondii responsible for family and TRAP family (containing TSR and type A domains) parasite spread throughout different host tissues. (Boucher and Bosch, 2015; Beeson et al., 2016) are responsible for Sporozoite: infective form of Plasmodium spp.; it is inoculated by the erythrocyte invasion. Microneme DBLs (only found in Plasmodium insect vector in the host dermis. Sporozoites travel along host body spp.) recognize different receptors, such as sialylated glycoproteins bloodstream and invade hepatocytes in the liver originating the exoerythrocytic phase of malaria. (e.g. glycophorins) by erythrocyte-binding antigens (EBAs) Merozoite: infective form of Plasmodium spp.; it is first delivered by the (Cowman et al., 2017) or specific proteins, as for the binding of infected hepatocyte, reaches the bloodstream and then invades host the DBP in P. vivax to Duffy antigen (also known as ACKR1) erythrocytes. After replication inside erythrocytes, more merozoites are located on red blood cells (Adams et al., 1992; Batchelor et al., cyclically delivered, giving rise to the erythrocytic phase of malaria. 2014). Proteins of the rhoptry RBL family in P. falciparum mediate Trypomastigote: infective form of T. cruzi present in the feces of the a sialic acid-independent invasion; for example, Rh4 binds to insect vector; it is deposited in host skin during vector blood meal. This form is also found in the mammalian host bloodstream after parasite complement receptor 1 (CR1) and Rh5 to basigin, which are both replication as the amastigote form and is responsible for parasite spread present on the erythrocyte PM (Crosnier et al., 2011; Tham et al., throughout different host tissues. 2010). During invasion, P. falciparum (Pf)Rh5 forms a complex Amastigote: intracellular replicative form of T. cruzi or Leishmania spp. with two other proteins, Rh5-interacting protein (PfRipr) and found in host cell cytosol or whiting vacuoles, respectively. cysteine-rich protective antigen (CyRPA) (Chen et al., 2011; Dreyer Epimastigote: extracellular replicative form of T. cruzi found in the et al., 2012). This possibly mediates the formation of a pore through intestine of the insect vector. This form originates the trypomastigote the membranes of both merozoite and erythrocyte, which allows form within the insect vector. 2+ Promastigote: infective form of Leishmania spp.; it is inoculated by the the release of Ca or the injection of parasite proteins inside insect vector in host dermis during vector blood meal. erythrocytes (Volz et al., 2016; Weiss et al., 2015). Owing to its essential role in basigin binding during erythrocyte invasion by merozoites, Rh5 is a leading candidate antigen for vaccine development against P. falciparum infection (Payne et al., 2017). 2015; Goel et al., 2003), as well as to heparin proteoglycans (Boyle Microneme TRAP family members, such as those relevant for et al., 2010). T. gondii, also have a role in Plasmodium spp. zoite motility and are The key factors in the recognition and strong adhesion of important for skin-to-liver migration and cell invasion (Morahan apicomplexan to the host cell surface are proteins secreted by the et al., 2009; Moreira et al., 2008; Sultan et al., 1997). Compared to micronemes (and also by rhoptries in Plasmodium), the microneme T. gondii, TRAP members in Plasmodium spp. are more diverse, adhesive proteins (MICs). Micronemal proteins are the first to be and specific TRAP proteins are expressed in the different zoite secreted (Box 2) and are essential for both host cell adhesion and stages (Morahan et al., 2009). A recent study has shown that the parasite motility and invasion. Secreted MICs are not resident in the human cell-surface integrin αvβ3 could act as a receptor for TRAP PM, but the presence of hydrophobic domains on some MICs in sporozoites (Dundas et al., 2018). In merozoites, MTRAP binds allows an individual MIC or their complexes to be embedded in the to semaphorin-7A at the erythrocyte surface (Bartholdson et al., parasite membrane (Huynh et al., 2003; Meissner et al., 2002; Reiss 2012), and is important not only for motility and invasion, but also et al., 2001; Sheiner et al., 2010). MICs contain either a single for gamete egress from erythrocytes inside the insect vector adhesive domain or a variety of combinations. Among the adhesive (Bargieri et al., 2016; Baum et al., 2006). domains found in T. gondii MICs are the microneme repeat domain (MAR family) (Blumenschein et al., 2007), the type A and TSR The formation of the moving junction domains (found in the TRAP family) (Wan et al., 1997) and Apple/ Rhoptry proteins are subcompartmentalized into two distinct PAN domains (Brecht et al., 2001), as well as the chitin-binding-like regions with different functions during the invasion process (CBL) domain (Céredè et al., 2002). Whereas some domains, such (Counihan et al., 2013); neck proteins act in the formation of a as TSR, are conserved among many species of Apicomplexa, others structure called the moving junction (MJ) or tight junction (Besteiro are more restricted, such as Apple and MAR, which are specific et al., 2011), whereas proteins from the bulb have a broad spectrum for coccidians (Friedrich et al., 2010). Binding assays have shown of functions, such as in cell adhesion (Beeson et al., 2016), that the MAR domain in T. gondii (Tg)MIC1 and TgMIC13 parasitophorous vacuole (PV) formation (Ghosh et al., 2017) and selectively interacts with sialylated oligosaccharides, which are immune evasion (Hakimi et al., 2017). The existence of a MJ was common terminal carbohydrates of the glycocalyx in vertebrate first described by electron microscopy observations of Plasmodium cells (Friedrich et al., 2010). In addition, the TSR domain of knowlesi and is morphologically characterized by the close TgMIC2 is important for T. gondii motility and cell invasion apposition between the parasite and host cell PMs, associated (Huynh and Carruthers, 2006). Indeed, the interaction of TgMIC2 with an electron-dense region right below the host cell PM (Aikawa type A domain with intercellular adhesion molecule 1 (ICAM-1) is et al., 1978) (Fig. 1A). MJs are conserved in T. gondii (Fig. 1B) and possibly involved in parasite migration across polarized epithelial Plasmodium spp., and their main molecular components are the cells (Barragan et al., 2005). rhoptry neck proteins (RON)2, RON4 and RON5, as well as the In Plasmodium sporozoites, the micronemal proteins P36 and microneme protein apical membrane antigen 1 (AMA1), a

P52 of 6-cysteine domain proteins (the s48/s45 family) are essential transmembrane protein secreted at the apical cap of the zoite Journal of Cell Science

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(Alexander et al., 2005; Lebrun et al., 2005; Bradley et al., 2005; flagellar bag, this mitochondrion presents a condensation of its Besteiro et al., 2009; Curtidor et al., 2011; Narum et al., 2008). DNA, named the kinetoplast, which can be easily visualized in Additionally, RON4L1 and RON8 are found in T. gondii MJs stained cells. The order Kinetoplastida is composed of several parasite (Guérin et al., 2017; Straub et al., 2009), and RON2 is inserted into protists of different genera that infect several animals, including the host PM, acting as a receptor for AMA1, which is located on the humans. Important pathogenic species of this order are Trypanosoma parasite apex (Besteiro et al., 2009; Cao et al., 2009; Lamarque cruzi (the agent of Chagas disease) and different species of the genus et al., 2011; Richard et al., 2010). Thus, zoites provide their own Leishmania (agents of leishmaniasis) (Fig. 2; Box 1). interacting ligand on the host cell to mediate the invasion process. The interaction between RON2 and AMA1 is of high affinity; thus, Cell invasion by Trypanosoma cruzi once the MJ is formed, zoites are committed to invasion (Delgadillo Infection by T. cruzi starts with the binding of infective et al., 2016; Srinivasan et al., 2011), which then occurs within trypomastigote forms (see Glossary) to the host cell PM, which seconds (Morisaki et al., 1995). Whereas RON2 and AMA1 are induces signaling pathways that culminate in its internalization into localized on the interface between the zoite and host cell surfaces, an endocytic vacuole (reviewed by Andrade and Andrews, 2004; RON4, RON4L1, RON5 and RON8 are localized in the host Fernandes and Andrews, 2012). Early studies of T. cruzi–host-cell cytoplasm (Beck et al., 2014; Guérin et al., 2017; Straub et al., interaction demonstrated that, soon after invasion, T. cruzi resides in 2011). During MJ assembly, cytosolic RONs in T. gondii cooperate an acidic vacuole that contains lysosomal markers (Milder and in recruiting host adaptor proteins, such as CIN85 (also known as Kloetzel, 1980; de Carvalho and de Souza, 1989). However, because SH3KBP1), CD2AP and the ESCRT-I components ALIX (also invasion is not only independent of actin filaments (Schenkman et al., known as PDCD6IP) and TSG101, which might facilitate the 1991), but also even facilitated by their disruption (Schenkman et al., physical interaction between the RON complex and the cortical 1991), phagocytosis can be excluded as a mechanism of parasite actin cytoskeleton (Guérin et al., 2017) (Fig. 1B). Furthermore, entry into host cells. This corroborates the fact that T. cruzi is able to secretion of toxofilin by T. gondii during invasion leads to invade any nucleated cell, including non-professional phagocytic disassembly of the actin meshwork at the site of entrance, cells (Fig. 2A,B), such as myocytes, which are actually the main permitting anchorage of the RON complex to a newly formed, target cells of the parasite during chronic infection in the vertebrate ring-shaped F-actin structure in the MJ region (Gonzalez et al., host (Calvet et al., 2012). 2009; Delorme-Walker et al., 2012). Such a ring-shaped F actin The attachment of T. cruzi provokes a rise in Ca2+ in the host cell structure was also observed at the site of MJ during Plasmodium cytosol, which signals the recruitment of host cell lysosomes to sporozoite invasion (Gonzalez et al., 2009). By contrast, an actin the parasite attachment site (Rodríguez et al., 1996) (Figs 2A, 4B). reorganization in erythrocytes linked to the MJ has not been Lysosomes then fuse with the host cell PM, releasing their content to observed during merozite invasion, but surprisingly, the presence of the external milieu. In doing so, they provide the membrane for the the cytoskeletal linker adducin was detected (Zuccala et al., 2016). nascent PV, leading to parasite internalization. After complete The association of cytosolic RON proteins with the cortical enveloping of the parasite, the PV, which is rich in lysosomal- cytoskeleton at the site of zoite entrance allows the MJ-anchored associated membrane proteins (LAMPs), is pinched off into the zoite to use the traction force of its actomyosin motor to make an cytoplasm through as-yet-unidentified mechanisms. The source of invagination in the host PM (Bichet et al., 2014; Gonzalez et al., the increased Ca2+ in the host cell cytosol is either intracellular 2009; Straub et al., 2011). During T. gondii invasion, the MJ stores, mobilized by signaling triggered by surface and secreted selectively excludes host transmembrane proteins from the forming proteins originating from the parasite, or from the extracellular PV membrane; this might prevent its fusion with the host lysosomes milieu, which can influx through parasite-induced micro-injuries (Bichet et al., 2014; Charron and Sibley, 2004; Mordue et al., 1999), inflicted on the host cell PM (Rodríguez et al., 1997; Rodríguez which would be deleterious to the parasite. Furthermore, during et al., 1999; Fernandes et al., 2011). The understanding of this entry T. gondii invasion, disassembly of the actin meshwork underlying mechanism came from studies of a basic physiological process used the PM, which is caused by the secretion of profilin during the initial by nucleated cells to repair PM wounds. Plasma membrane repair host–pathogen interaction, also acts in decreasing the resistance of (PMR) is driven by (1) Ca2+ influx, (2) lysosomal exocytosis and host cell membrane invagination (Delorme-Walker et al., 2012). (3) extensive Ca2+-dependent and actin-independent endocytosis Similarly, a cytoskeleton reorganization at erythrocyte entry site is (Bi et al., 1995; Togo et al., 2000; Reddy et al., 2001; Idone et al., also required for P. falciparum invasion (Dasgupta et al., 2014). 2008). During PMR, one of the enzymes secreted by the lysosomes, This process is regulated by Ca2+ signaling and phosphorylation acid sphingomyelinase (ASM), is able to trigger endocytosis of several erythrocyte cytoskeletal proteins, including β-spectrin, through the formation of ceramide at the cell surface, which PIEZO1 and band 3, leading to a destabilization of the cytoskeleton facilitates inward budding and thus endocytosis of the wounded (Fernandez-Pol et al., 2013; Wernimont et al., 2010; Zuccala et al., membrane (McIntosh et al., 1992; Brown and London, 2000; Tam 2016). After invasion, the newly formed PV pinches off from the et al., 2010). Based on these findings, Fernandes and co-workers PM through an as-yet elusive mechanism. Experiments using the proposed that T. cruzi subverts PMR for entry into the host cell dynamin inhibitor dynasore in T. gondii suggest that the host cell (Fernandes et al., 2011). Further evidence of PMR involvement dynamin participates in the pinching off of the PV (Caldas et al., in T. cruzi invasion comes from the observation that recently 2009). However, more recently, it has been shown that the pinch off internalized parasites are found in ceramide-rich vacuoles that are of the T. gondii PV is independent of the host cell, and appears to be formed by the action of ASM during parasite-induced PMR induced by a twisting motion of the parasite, which mechanically (Fernandes et al., 2011). mediates membrane scission and PV detaching (Pavlou et al., 2018). Several parasite surface proteins involved in invasion have been described (Ruiz et al., 1998; Caler et al., 1998; Scharfstein et al., Kinetoplastids – Trypanosoma cruzi and Leishmania spp. 2000; Neira et al., 2003; Kojin et al., 2016; Alves and Colli, 2008; Kinetoplastids are flagellated protozoans characterized by the reviewed by Maeda et al., 2012). Among them are the members of presence of a large and unique mitochondrion. Close to the the gp85/trans-sialidase (TS) super-family (gp82, gp90, gp85/TS, Journal of Cell Science

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A B

C D

Fig. 2. The kinetoplastids Trypanosoma cruzi and Leishmania spp. invade cells through both recruitment of host cell lysosomes and phagocytosis. (A) Fibroblast infection by T. cruzi trypomastigotes. The immunofluorescence microscopy image (L.O.A.) shows lysosomes (green), host cell and parasite nuclei, and the parasite kinetoplast (blue). The red arrow indicates an extracellular T. cruzi trypomastigote, while the white arrow points to a recently internalized parasite with lysosomes being recruited to the PV. The green arrow points to a parasite inside a PV that is covered with lysosomal markers. (B) A trypomastigote form of T. cruzi infecting an adipocyte host-cell (HC). Image republished with permission of the American Society for Biochemistry and Molecular Biology, from Combs et al., 2005; permission conveyed through the Copyright Clearance Center. (C) Immunofluorescence microscopy image of a Leishmania promastigote (red, stained by anti-LPG antibody; red arrow indicates the still extracellular parasite body) invading a fibroblast by its flagellar tip through the recruitment of host cell lysosomes (green). Host cell nuclei are stained by DAPI. Adapted with permission from Cavalcante-Costa et al. (2019). (D) Scanning electron-microscopy of a Leishmania promastigote being captured through phagocytosis by a macrophage. The white arrow points to the macrophage phagocytic cup and the black arrow to the parasite body. Adapted with permission from Zenian et al. (1979). gp30); of these, gp82 has been recently shown to be recognized by Cell invasion by Leishmania spp. the host-cell lysosomal protein LAMP-2, which may be present Histological tissue sections of mammalians hosts with leishmaniasis at low levels on the host-cell surface (Rodrigues et al., 2019). reveal that macrophages are the ultimate host cells for Leishmania Binding of gp82 to target cells induces lysosome spreading and spp. (Benchimol and de Souza, 1981; Berman et al., 1981; Brazil, exocytosis, culminating in parasite internalization (Martins et al., 1984; Davies et al., 1988). However, the circumstances under which 2011; Cortez et al., 2016). Two parasite enzymes have also been macrophages are invaded in vivo by promastigotes (see Glossary) implicated in the induction of Ca2+ signaling in host cells. are not completely understood, particularly with regard to the Oligopeptidase B appears to generate a product that is recognized molecular details. Nevertheless, in vitro, phagocytosis has been by a G-protein-coupled receptor (Tardieux et al., 1994; Burleigh and suggested as the main route of promastigote invasion, because, in Andrews, 1995), while the cysteine protease cruzipain cleaves host the absence of host cell actin polymerization, macrophage infection kininogen into bradykinin, which interacts with its classical is impaired (Akiyama and Haight, 1971; Alexander, 1975; Ardehali bradykinin receptor on the host (Scharfstein et al., 2000). Both et al., 1979; Zenian et al., 1979; Roy et al., 2014) (Fig. 4C). stimuli eventually converge in a pathway that involves Ca2+ In vitro, phagocytosis of promastigotes by macrophages (Fig. 2D) signaling-mediated events and host cell lysosome recruitment, appears to start only after two minutes of contact with parasites resulting in parasite internalization (Rodríguez et al., 1999; Caler (Aikawa et al., 1982). It is remarkable that, during the first moments et al., 1998). T. cruzi trypomastigotes can also interact with host of contact, 90% of promastigotes are attached, with low affinity, extracellular matrix components, which appears to facilitate their to macrophages (Uezato et al., 2005) through their flagellar tip contact with and invasion into host cells (Santana et al., 1997; Alves (Aikawa et al., 1982), suggesting a role for this structure in triggering and Colli, 2008; Nde et al., 2012). phagocytosis. It is worth pointing out that promastigotes generally It is important to mention that amastigotes (see Glossary), which move in the direction of the flagellum, an anterior structure (Krüger can be released extracellularly are also able to invade host cells and Engstler, 2015), that has been proposed to have a role in sensing (Fernandes et al., 2013). In contrast to trypomastigotes, however, the environment (Rotureau et al., 2009), probably as it is the first invasion of amastigotes into host cells occurs through an actin structure to encounter a host cell. After five minutes, however, polymerization-dependent pathway (Fernandes et al., 2013), and parasites are tightly bound to macrophages and phagocytosed with no amastigotes are also able to induce phagocytosis in non- preferred orientation (Aikawa et al., 1982; Uezato et al., 2005). The professional phagocytic cells (Ferreira et al., 2019). vast majority of phagocytosed promastigotes clearly localize inside Journal of Cell Science

6 REVIEW Journal of Cell Science (2020) 133, jcs232488. doi:10.1242/jcs.232488 phagosomes (Courret et al., 2002), which promptly fuse with and heparan sulfate (Maciej-Hulme et al., 2018) have also been lysosomes (James et al., 2006; Courret et al., 2002); this creates an implicated in the internalization of promastigotes by macrophages. appropriate milieu for their fully differentiation into amastigotes (see Moreover, and importantly, even though macrophages are the Glossary), and subsequent replication (Alexander and Vickerman, final and main destination of parasites during the chronic phase of 1975; Chang and Dwyer, 1976; Moradin and Descoteaux, 2012). leishmaniasis, and may actually be directly infected by Several macrophage receptors have been described to mediate promastigotes in vivo (Peters et al., 2008), neutrophils are the parasite adherence and subsequent phagocytosis signaling (Mosser major cells to capture promastigote during the initial phase of and Rosenthal, 1993; Lefevrè et al., 2013; Polando et al., 2018, infection (Peters et al., 2008). In this case, macrophages may also 2018; Stafford et al., 2002; reviewed by Naderer et al., 2005 and become secondarily infected by ingesting Leishmania-containing Ueno and Wilson, 2012; Chauhan et al., 2017; von Stebut and apoptotic bodies after neutrophil death. This appears to be crucial Tenzer, 2018). One such type of receptor, the endocytic mannose for the survival of parasites and the outcome of infection (Afonso receptors (MRs) in macrophages, which are pattern recognition et al., 2008) and has been earlier referred to as the ‘Trojan horse receptors (PRRs), were previously considered to be necessary for strategy’ (Laskay et al., 2003). attachment and phagocytosis of L. donovani and L. infantum Dendritic cells (DCs) are also targets for Leishmania spp. promastigotes by macrophages (Blackwell, 1985; Wilson and invasion. However, invasion of DCs is reported to occur mostly at Pearson, 1986; Chakraborty et al., 1998; Ueno et al., 2009; later phases of the disease when susceptible promastigotes had Polando et al., 2018). However, more recently, their roles have been either died through complement-mediated lysis or transformed into contested based on several lines of evidence. First, MR ligands can amastigotes inside macrophages, and when antibodies to parasites also inhibit the uptake of L. donovani and L. major in the absence of appear in sera. In fact, it has been shown that DCs preferentially take MRs (Akilov et al., 2007). Secondly, inhibition of parasite uptake up amastigotes, rather than promastigotes, requiring opsonization by MR ligands does not occur for all stages of promastigotes (Ueno through their receptor FcγR, which recognizes IgG (Guy and et al., 2009) or all species (Da Silva et al., 1989; Mosser and Belosevic, 1993; Peters et al., 1995; Kima et al., 2000). Handman, 1992), and, finally, MR-depleted C57BL/6 macrophages Much less is known about the direct invasion of amastigotes or do not differ in their infection rate compared to wild-type cells their transfer from one host cell to another, although this has been with regard to various aspects of the disease outcome, suggesting reported to occur via macrophage receptors for Fcγ or the existence of redundant infection receptors (Akilov et al., 2007). phosphatidylserine (Love et al., 1998; Kane and Mosser, 2000; One explanation for these conflicting findings may be that De Freitas Balanco et al., 2001; Wanderley et al., 2006), mannose-carrying ligands in promastigotes can also bind to other phagocytosis of infected apoptotic bodies (Peters et al., 2008) or C-type lectin receptors (McGreal et al., 2005) and Toll-like receptor by direct cell-to-cell transfer of PV extrusions (Real et al., 2014). (TLR) family members (Roeder et al., 2004). Another PRR, TLR-2, Although the inhibition of phagocytosis mostly blocks invasion has also been implicated in the binding and internalization of of macrophages by promastigotes, a small number of parasites can L. major by BALB/c macrophages through its interaction with still be found inside these cells (Roy et al., 2014; Lewis, 1974; lipophosphoglycan (LPG) (Srivastava et al., 2013), a highly Aikawa et al., 1982), indicating that Leishmania can invade prevalent surface glycoconjugate in promastigotes. macrophages by routes other than classical phagocytosis. In fact, In addition to PRRs, it is well known that complement receptor 3 it has long been described that promastigotes can also invade non- (CR3; comprising integrin αM and integrin β2) (Russell and phagocytic cells (Bogdan et al., 2000; Minero et al., 2004; Holbrook Wright, 1988), which also binds to LPG (Van Strijp et al., 1993; and Palczuk, 1975; Schwartzman and Pearson, 1985). Recent work Talamás-Rohana et al., 1990) and fibronectin receptors (Soteriadou from our group has unveiled a new non-phagocytic route of et al., 1992; Rizvi et al., 1988; Brittingham et al., 1999), bind to promastigote invasion without any involvement of the host cell gp63, a major leishmanial GPI-anchored surface metalloproteinase. cytoskeleton, thereby providing definitive evidence for an entry LPG and gp63 have been reported to be important for internalization mechanism other than phagocytosis (Cavalcante-Costa et al., 2019). of promastigotes by macrophages (Brittingham et al., 1999; This pathway involves the Ca2+-dependent recruitment and Gorocica et al., 2000). However, other results, which showed that exocytosis of host cell lysosomes to the parasite invasion site, LPG-deficient (Ilg, 2000; Späth et al., 2003) or gp63-deficient where they instantly fuse with the PM, much like in the invasion (Joshi et al., 2002) mutant promastigotes are as efficient as wild- mechanism described above for T. cruzi. This creates a niche for the type parasites in invading macrophages in vitro or infecting mice, internalization of promastigotes, which most often occurs through indicate that these molecules are not critical for invasion, at least for the flagella (Cavalcante-Costa et al., 2019) (Fig. 2C). It is possible, the Leishmania species studied. therefore, to speculate that, in vivo, Leishmania does not solely rely Although, the direct binding of promastigotes to macrophages that on being captured by phagocytes to establish infection, and that occurs in vitro could also take place in vivo, the latter scenario is different infected cell types of the dermis, as already reported by considerably more complex. Thus, it is very likely that opsonization other groups (Locksley et al., 1988; Bogdan et al., 2000), could act may have a significant role in parasite uptake, as they most probably as Trojan horses. interact first with other host molecules, which would facilitate phagocytosis. In this regard, a major molecule is complement Microsporidians component 3 (C3), which, in vitro, mainly binds to gp63 and LPG Microsporidians have long been considered as primitive protozoans after complement activation (Podinovskaia and Descoteaux, 2015; with which they share some morphological similarities, but more Sacks, 1992). The generated fragment C3b has been reported to be accurate phylogenetic analysis have shown that they are, in fact, essential for promastigote entry into macrophages through binding to related to fungi (Weiss et al., 1999; James et al., 2006). These CR1 (Da Silva et al., 1989), while iC3b, a product of C3b cleaved by parasites were described by Louis Pasteur as a plague that decimated gp63, opsonizes the parasites by binding to CR3 (Brittingham et al., silkworms with a huge impact in silk industry in France, almost 1995). Other opsonizing molecules, including fibronectin (Vannier- 150 years ago (Pasteur and Pasteur, 1870). Microsporidians are

Santos et al., 1991), C-reactive protein (Bodman-Smith et al., 2002) spread through parasite spores, which can infect not only several Journal of Cell Science

7 REVIEW Journal of Cell Science (2020) 133, jcs232488. doi:10.1242/jcs.232488 animals of economic importance, but also humans, being important shown to reduce infections in vitro, making this protein a promising pathogens. The phylum Microsporidia is composed by over 140 new target for drug and vaccine development (Han et al., 2017; genera, but only eight have been characterized as causative of reviewed by Han and Weiss, 2017). human microsporidiosis: Enterocytozoon, Encephalytozoon, Even though microsporidians have been known about and studied Pleistophora, Trachipleistophora, Vittaforma, Brachiola, Septata for more than a century, essential features, such as the mechanism of and Nosema (Weiss, 2000). In humans, these parasites are able to direct injection into the cytoplasm and the endocytosis of the infect almost every organ system causing asymptomatic, chronic or infective sporoplasm by the host cell still remain a matter of lethal infections, depending on the immunological status of the discussion. Interestingly, it has been shown that if the spore is patient (Weber et al., 2000, 1994) (Box 1). phagocytosed by macrophages, the extrusion of the polar tube, typically termed germination, occurs from within the PV, leading to Cell invasion by microsporidians the perforation of the vacuolar membrane and the delivery of the Microsporidian spores are rigid and round, and range from 1 to infectious sporoplasm directly into the cytosol (Franzen, 2005); this 12 µm in size, depending on the species; they can be divided in three constitutes a means to evade the deleterious conditions within basic morphological structures: the spore, the sporoplasm, which fusogenic vacuoles. It is also interesting to note that microsporidians is the parasite body, and the invasion apparatus (Han and Weiss, are able to replicate inside PVs, which are modified by the parasite 2017). The spore wall is mainly composed of chitin and to escape lysosomal acidification and to allow nutrient uptake from glycoproteins (Han and Weiss, 2017). The most important family the cytoplasm (Rönnebäumer et al., 2008). Indeed, the membrane of of proteins that form the spore wall are named spore wall proteins the PV that contains Encephalitozoon cuniculi has been shown to (SWPs), and these are essential for the formation of the spore but originate from the host cell PM (Fig. 3C), as demonstrated by the also participate in the adhesion of the spore to host cell PM before addition of dyes to the host cell PM, which were found in the PV the formation of the invading apparatus, named the polar tube membrane shortly after cell invasion (Rönnebäumer et al., 2008). (recently reviewed by Yang et al., 2018) (Figs 3, 4D). The polar tube As PTP4 localizes to the tip of the polar tube and is able to interact is a coiled structure, which is extruded in a fraction of a second, as a with the host cell PM, Han et al. have proposed a model, in which result of osmotic changes inside the spore (Fig. 3A,B). It can be as PTP4 together with PTP1 helps forming the invagination of the host long as 500 µm, depending on the species, with a narrow diameter cell PM that excludes the extracellular environment, thus creating of between 0.1 and 0.2 µm, through which the sporoplasm travels to a microenvironment that is protected from host innate immune infect the host cell (Weidner, 1976; Han and Weiss, 2017). Polar responses, thereby facilitating invasion (Han et al., 2017). However, tube proteins (PTPs), which form the polar tube, are the main factors the machinery for pinching off and fission, which subsequently involved in invasion. At least five PTPs have been described in detaches the nascent membrane of the PV is still unknown. It has microsporidians, with PTP1, PTP2, PTP3 and PTP5 found evenly been hypothesized that invasion could also involve the clathrin distributed throughout the polar tube, whereas PTP4 is specifically mediated-endocytosis machinery (Han et al., 2017), or host cell located to the polar tube tip, in close interaction with host cell PM at actin polymerization (Foucault and Drancourt, 2000). Although the infection synapsis (Han et al., 2017). PTP4 binds to transferrin these obligatory intracellular parasites have the smallest genome receptor 1 (TfR1, also known as TFRC) on the PM of mammalian among eukaryotes, they have evolved one of the most remarkable cells, and blocking this binding with specific antibodies has been machinery to invade host cells. In this context, a better

Fig. 3. Microsporidians invade cells through the formation of a polar tube. (A,B) Correlative light and electron microscopy (CLEM) analysis of Encephalitozoon hellem infection. The fluorescence image and scanning electron microscopy (SEM) image of the same site were taken sequentially. The fluorescence signal showing the labeling of polar tube protein 1 (PTP1, red; all over the tube) and polar tube protein 4 (PTP4, green; tube tip). The enlargement in B shows the droplet of released sporoplasm (SP). (C) Transmission electron microscopy (TEM) data demonstrating that the polar tube is surrounded with host cell membrane (black arrows) at the site of infection. PT, polar tube; PM, plasma membrane. Scale bars: 2 µm (A,B, main images); 1 µm (B, enlargement); 400 nm (C). Adapted from Han et al. (2017), where it was published under a CC BY 4.0 license. Journal of Cell Science

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A Toxoplasma gondii B Trypanosoma cruzi Fig. 4. Overview over the cell invasion Plasmodium spp. mechanisms employed by apicomplexans, kinetoplastides and microsporidians to entry host cells. (A) (1) Apicomplexans (e.g. T. gondii and 1 Plasmodium spp.) first weakly interact with the host Ca2+ cell PM using their resident-GPI anchored proteins. At the same time, the strong interaction provided by 1 microneme-secreted protein (MICs) leads to zoite Moving junction- Lysosome- 2 re-orientation. (2) With the apical complex facing the 2 mediated entry mediated host cell PM, proteins secreted by the rhoptries entry interact with host cell proteins and (3) form the moving junction (see Fig. 1), which provides the 3 3 force that drives parasites into the host cell cytoplasm. Inside host cells, parasites reside in a non-fusogenic vacuole and so avoid lysosomal 4 fusion. (B) T. cruzi invades non-phagocytic host cells by inducing both (1) extracellular Ca2+ influx through 4 parasite-induced PM wounds and the release of intracellularly stored Ca2+. (2) This leads to the 5 recruitment of host cell lysosomes that function as Ca2+-sensitive exocytic vesicles, which donate membranes to the nascent PV. (3) Inside host cells, Phagocytosis the vacuole keeps fusing with host cell lysosomes, 2 1 leading to (4) parasites escaping into the host cell cytosol where they replicate as round-shaped 3 2 amastigotes. (C) Leishmania spp. are captured by or phagocytes through either (1) actin-mediated phagocytosis or (2) by non-phagocytic cells through Ca2+-dependent recruitment of host cell lysosomes that donate membranes to the nascent PV. This Polar tube- Lysosome- process appears to be the same as infection by Ca2+ mediated entry mediated T. cruzi trypromastigotes, indicating that the entry 1 1 underlying mechanisms are conserved in these kinetoplastides. After invasion, Leishmania spp. live and replicate inside acidic vacuoles that fuse with Host cell host cell lysosomes. (D) Microsporidian spores bind to host cells with their spore wall and extrude their D Microsporidians C Leishmania spp. polar tube, which creates an invagination in the host cell PM. The infective sporoplasm that harbors the spore travels along the polar tube lumen and is delivered at the infection site. After internalization, Key MICs/AMA-1 protein Host cell proteins Host cell lysosomes the parasite lives and replicates inside non- fusogenic vacuoles and so avoids lysosomal fusion. Rhoptry proteins Moving junction Host cell actin filaments understanding of the proteins involved in spore adhesion to host cell occur without the initial adhesion of the parasite to the host cell, the PM (SWPs) and the ones involved in the functioning of the polar discovery of the underlying molecules is also crucial. Another point tube (PTPs), as well as the discovery of the molecular actors that merits additional discussion is how infective Leishmania involved in PV pinch-off are crucial to unveil parasite entry process. promastigotes that are delivered by sand flies actually reach macrophages. We know today, for instance, that after infection of Concluding remarks mice with promastigotes, macrophages are not necessarily the first Even though the parasites discussed here have been known and cells that are invaded by the parasite (Peters et al., 2008), and that the studied for decades, some key issues of their biology and the details indirect infection of macrophages through phagocytosis of of the mechanism by which they invade host cells remain elusive. amastigotes, expelled from infected cells from PVs or as apoptotic Many questions still remain open with regard to the invasion bodies, or other mechanisms, are more likely to occur. Moreover, processes presented here. For instance, how exactly do nascent PVs most conditions of in vitro infections, which are used to study the pinch off from the PM and which membrane fission machinery is cell invasion process, do not recapitulate the in vivo situation, for responsible for this crucial invasion step? Is membrane fission instance, the use of fresh serum as a source of complement, because driven by the parasite itself, or does it require the recruitment of C3 opsonization of promastigotes, which is likely to occur in vivo, host cell molecules? Some attempts to solve this question may be strongly increases their ability to infect macrophages. Hence, still controversial. For instance, results drawn from inhibition findings based solely on in vitro infection of macrophages by experiments with dynasore might be ambiguous in inferring that promastigotes should be interpreted with caution. Thus, more dynamin participates in this process owing to the cytotoxic and off- physiologically relevant approaches, such as in situ visualization of target effects of this drug (Preta et al., 2015). Therefore, more mice infections by intravital microscopy, using labeled cells and advanced approaches, such as the use of CRISPR/Cas9 or RNAi molecules, are needed to reach a better understanding of the silencing to target candidates, such as dynamin itself, as well as invasion processes by these intracellular parasites. Similarly, other host cell proteins involved in membrane fission are anticipated experiments aiming to avoid off-target drug effects and to more to provide more reliable results. Importantly, as invasion does not closely reflect the processes occurring in nature would certainly be Journal of Cell Science

9 REVIEW Journal of Cell Science (2020) 133, jcs232488. doi:10.1242/jcs.232488 helpful in making appropriate choices for molecular targets used in Alves, M. J. M. and Colli, W. (2008). Role of the gp85/trans-sialidase superfamily of drug discovery and vaccine development. glycoproteins in the interaction of trypanosoma cruzi with host structures. Subcell. Biochem. 47, 58-69. doi:10.1007/978-0-387-78267-6_4 Finally, some features of the invasion processes are common Andrade, L. O. and Andrews, N. W. (2004). Lysosomal fusion is essential for the among phylogenetically-related parasites. For example, cell retention of Trypanosoma cruzi inside host cells. J. Exp. Med. 200, 1135-1143. invasion by Plasmodium spp. and Toxoplasma gondii zoites may doi:10.1084/jem.20041408 Araújo, C. A. C., Waniek, P. J. and Jansen, A. M. (2009). An overview of Chagas differ in terms of the specific host cell invaded, initial adhesion disease and the role of triatomines on its distribution in Brazil. Vector Borne molecules or proteins used for the assembly of the MJ, but, Zoonotic Dis. 9, 227-234. doi:10.1089/vbz.2008.0185 ultimately, the same invading machinery is formed. Similarly, we Ardehali, S. M., Khoubyar, K. and Rezai, H. R. (1979). Studies on the effect of the anti-phagocytic agent cytochalasin B on Leishmania-macrophage interaction. now know that the infective stages of both T. cruzi and Leishmania Acta Trop. 36, 15-21. are able to induce cell invasion by subverting PMR-induced Arredondo, S. A., Swearingen, K. E., Martinson, T., Steel, R., Dankwa, D. A., endocytosis (Fig. 4). Thus, building on established knowledge, Harupa, A., Camargo, N., Betz, W., Vigdorovich, V., Oliver, B. G. et al. (2018). extrapolation of models and mechanisms from one species could The micronemal plasmodium proteins P36 and P52 act in concert to establish the replication-permissive compartment within infected hepatocytes. Front. Cell. help to narrow knowledge gaps in closely related microorganisms, Infect. Microbiol. 8, 413. doi:10.3389/fcimb.2018.00413 thereby accelerating new discoveries and guiding the proposal of Azzouz, N., Kamena, F., Laurino, P., Kikkeri, R., Mercier, C., Cesbron-Delauw, new approaches to better understand the biology of these parasites M.-F., Dubremetz, J.-F., De Cola, L. and Seeberger, P. H. (2013). Toxoplasma gondii secretory proteins bind to sulfated heparin structures. Glycobiology 23, and the diseases they cause. 106-120. doi:10.1093/glycob/cws134 Baldwin, M. R., Li, X., Hanada, T., Liu, S. C. and Chishti, A. H. (2015). Merozoite Acknowledgements surface protein 1 recognition of host glycophorin a mediates malaria parasite T. C.-G. and É. S. M.-D. would like to thank Professors Égler Chiari and Lúcia Maria invasion of red blood cells. Blood 125, 2704-2711. doi:10.1182/blood-2014-11- C. Galvão from Universidade Federal de Minas Gerais, Brazil, for all their support to 611707 our research groups and dedicate this review to their important accomplishments in Bargieri, D. Y., Thiberge, S., Tay, C. L., Carey, A. F., Rantz, A., Hischen, F., the field of protozoology, notably for their studies with Trypanosoma cruzi. We also Lorthiois, A., Straschil, U., Singh, P., Singh, S. et al. (2016). Plasmodium acknowledge the Departamento de Parasitologia, Universidade Federal de Minas Merozoite TRAP Family Protein Is Essential for Vacuole Membrane Disruption Gerais, for all its support during the establishment of our research groups and and Gamete Egress from Erythrocytes. Cell Host Microbe 20, 618-630. doi:10. laboratories. Thiago Castro-Gomes also acknowledges Professor Norma Windsor 1016/j.chom.2016.10.015 Andrews from the University of Maryland, USA, who first described the lysosome- Barragan, A., Brossier, F. and Sibley, L. D. (2005). Transepithelial migration of dependent mechanism of cell entry in Trypanosoma cruzi, for all her support and Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell. Microbiol. 7, 561-568. doi:10.1111/ shared knowledge. j.1462-5822.2005.00486.x Bartholdson, S. J., Bustamante, L. Y., Crosnier, C., Johnson, S., Lea, S., Competing interests Rayner, J. C. and Wright, G. J. (2012). Semaphorin-7A is an erythrocyte receptor The authors declare no competing or financial interests. for P. falciparum merozoite-specific TRAP homolog, MTRAP. PLoS Pathog. 8. doi:10.1371/journal.ppat.1003031 Funding Batchelor, J. D., Malpede, B. M., Omattage, N. S., DeKoster, G. T., Henzler- Our work in this field is supported by FAPEMIG (Fundação de Amparo àPesquisa Wildman, K. A. and Tolia, N. H. (2014). 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