cell invasion by intracellular parasites the many roads to ... · msp1 is proteolytically processed...

REVIEW SUBJECT COLLECTION: CELL BIOLOGY AND DISEASE

Cell invasion by intracellular parasites – the many roadsto infectionMaria Fátima Horta1, Luciana Oliveira Andrade2, Érica Santos Martins-Duarte3 and Thiago Castro-Gomes3,*

ABSTRACTIntracellular parasites from the genera Toxoplasma, Plasmodium,Trypanosoma, Leishmania and from the phylum Microsporidia are,respectively, the causative agents of toxoplasmosis, malaria, Chagasdisease, leishmaniasis andmicrosporidiosis, illnesses that kill millionsof people around the globe. Crossing the host cell plasma membrane(PM) is an obstacle these parasites must overcome to establishthemselves intracellularly and so cause diseases. Themechanisms ofcell invasion are quite diverse and include (1) formation of movingjunctions that drive parasites into host cells, as for the protozoansToxoplasma gondii andPlasmodium spp., (2) subversion of endocyticpathways used by the host cell to repair PM, as for Trypanosoma cruziand Leishmania, (3) induction of phagocytosis as for Leishmania or(4) endocytosis of parasites inducedbyspecialized structures, such asthe polar tubes present in microsporidian species. Understanding theearly steps of cell entry is essential for the development of vaccinesand drugs for the prevention or treatment of these diseases, and thusenormous research efforts have been made to unveil their underlyingbiological mechanisms. This Review will focus on these mechanismsand the factors involved, with an emphasis on the recent insights intothe cell biology of invasion by these pathogens.

KEY WORDS: Host cell invasion, Plasmodium spp., Toxoplasmagondii, Trypanosoma cruzi, Microsporidia

IntroductionEvolving a way to invade their host cells is the first obligatorychallenge for intracellular parasites. In order to establishintracellular life, these microorganisms employ numerousstrategies to overcome the barrier that is imposed by the host cellplasma membrane (PM) and cytoskeleton. Small intracellularpathogens, such as bacteria and viruses, can be endocytosedthrough natural and abundant PM invaginations, via clathrin-,caveolae- and flotillin-mediated endocytosis (Cossart and Helenius,2014). However, these small, nanoscale membrane invaginationscannot carry larger intracellular parasites, such as protozoans. Theinternalization of these pathogens can be achieved instead throughseveral other routes. Larger pathogens can actively drive cell entrythrough the formation of a specialized cell machinery, such as themoving junction that is present in the apicomplexans Plasmodiumspp. (Aikawa et al., 1978) and Toxoplasma gondii (Mordue et al.,1999). Invasion can also occur through the subversion of

physiological host cell processes, such as lysosome-triggeredCa2+-dependent endocytosis, which is used by all nucleated cellsto repair wounded PMs (Corrotte and Castro-Gomes, 2019); thispathway has previously shown to occur for Trypanosoma cruzi(Rodríguez et al., 1996; Fernandes et al., 2011), and also morerecently by our lab for Leishmania amazonensis (Cavalcante-Costaet al., 2019). Large parasites can be equally phagocytosed, if theyare able to resist phagocytic degradation and live within phagocyticcells, such as protozoans of the genus Leishmania (Zenian et al.,1979). Finally, internalization can also be induced through theformation of a specialized structure that directs the parasite towardsthe PM, as for microsporidians, for example, Encephalitozoon spp.(Xu andWeiss, 2005). Understanding the initial interaction betweena host cell and an intracellular parasite is essential, as the moleculardeterminants involved could be exploited as vaccine targets to helpblock the infection from the onset. This Review will focus on thecellular and molecular determinants that mediate host cell invasionby the above parasites paying special attention to the initial steps.

Apicomplexans – Plasmodium spp. and Toxoplasma gondiiApicomplexans are obligate intracellular parasites characterized bythe presence of an apical complex, a cellular structure crucial for cellinvasion composed of cytoskeletal components and three secretoryorganelles, namely, micronemes, rhoptries and dense granules (Adlet al., 2018). They also lack flagella, cilia or pseudopods, thusrelying on a specialized intracellular machinery that allows them tomove when attached to substrates or the host cell PM. Toxoplasmagondii (agent of toxoplasmosis) and Plasmodium spp. (agents ofmalaria) are included within this phylum (Fig. 1; Box 1).

Cell invasion by apicomplexansOwing to their inability to undergo cell division and limited viabilityin extracellular environments, apicomplexans have evolved a uniqueand extremely effective mechanism for invading a host cell, which isdependent on gliding motility and coordinated secretion of proteinscontained in their apical secretory organelles (Box 2) (Carruthers andSibley, 1997). In the initial moments of interaction with the host cell,the apicomplexan zoite (see Glossary) moves laterally on its surfaceuntil it encounters an ideal receptor that triggers host cell invasion;this then leads to an apical re-orientation of the zoite, which ismediated by microneme adhesins and is followed by the secretion ofrhoptry proteins into host cell cytoplasm through a transient pore inthe host cell PM (Carruthers and Boothroyd, 2007;Weiss et al., 2015;Dubremetz, 2007) (see Fig. 4A).

In T. gondii, the initial lateral weak interaction with hostcell membrane is mediated by glycosylphosphatidylinositol (GPI)-anchored proteins of the surface antigen glycoprotein (SAG)-relatedsequence (SRS) superfamily, which are resident on the parasite PM(Dzierszinski et al., 2000; Manger et al., 1998;Wasmuth et al., 2012).SRS genes are differently expressed according to lineage orevolutionary stage, and are involved in a wide range of functions,

1Departamento de Bioquıḿica e Imunologia, Instituto de Ciências Biológicas,Universidade Federal de Minas Gerais, Belo Horizonte, CEP 31270-901, Brazil.2Departamento de Morfologia, Instituto de Ciências Biológicas, UniversidadeFederal de Minas Gerais, Belo Horizonte, CEP 31270-901, Brazil. 3Departamentode Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de MinasGerais, Belo Horizonte, CEP 31270-901, Brazil.

*Author for correspondence ([email protected])

T.C., 0000-0003-1564-4645

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

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https://jcs.biologists.org/collection/cell_biology_diseasemailto:[email protected]://orcid.org/0000-0003-1564-4645

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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 merozoiteshows 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–merozoiteattachment site showing the thickened erythrocyte membrane. Right, an advanced stage of erythrocyte (E) entry by a merozoite (Mz). Note a junctionalattachment (labeled C) at each side of the entry orifice. Republished with permission of Rockefeller University Press from Aikawa et al., 1978; permissionconveyed through the Copyright Clearance Center. (B) Schematic representation of cell binding and formation of the moving junction during host cell invasionby the apicomplexan parasite Toxoplasma gondii. (1) Rhoptry secretion. After re-orientation, the parasite apical complex faces the host cell PM. A localloosening 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 amulti-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 enlargementshows a schematic representation of the moving junction. Rhoptry proteins (RON2, RON4, RON5 and RON8) form a molecular complex with host cellproteins of the ESCRT family (ALIX, TSG101, CIN85 and CD2AP); this anchors the parasite to the host cell actin cytoskeleton, while the extracellular domainof RON2 binds to AMA1 present on parasite PM. Inside the parasite, the cytosolic domain of AMA1 binds to a parasite actin-myosin motor through theglideosome-associated connector (GAC), while the rhomboid protease ROM4 successively disengages the complex to allow parasite moving. IMC, innermembrane complex. See also Box 2.

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including cell adhesion (Dzierszinski et al., 2000; Kim et al., 2007;Leal-Sena et al., 2018; Tomita et al., 2018; Wasmuth et al., 2012).Interaction of SRS proteins with the host cell or substrates is,however, weak, possibly allowing the parasite to move laterally alongtheir surface (Carruthers and Boothroyd, 2007). Disruption of theSAG3 gene, which codes for the SRS adhesive protein, reducesparasite adhesion to host cell and also decreases virulence of T. gondiito mice (Dzierszinski et al., 2000). It has also been shown that bothSAG3 and SAG2 interact with heparin sulfate proteoglycans (Jacquetet al., 2001; Zhang et al., 2019). SAG1 also interacts with sulfatedproteoglycans, as supported by structural studies and cell bindingassays (He et al., 2002; Azzouz et al., 2013).The initial interaction between Plasmodium zoites and host cells

also rely on parasite GPI-anchored proteins (Beeson et al., 2016),such as the circumsporozoite protein (CSP) (Yoshida et al., 1980)

and merozoite (see Glossary) surface proteins (MSPs) (reviewed byBeeson et al., 2016). CSP is highly conserved among Plasmodiumspecies and mediates sporozoite (see Glossary) migration frommammalian dermis to liver (Tewari et al., 2002). Upon reaching theliver, sporozoites switch from a migratory to an invasive mode.Sporozoite binding to heparan sulfate proteoglycans (HSPGs) thatare expressed by hepatic stellate cells culminates in the secretion ofcysteine proteases by the parasite (Coppi et al., 2005; Frevert et al.,1993; Pinzon-Ortiz et al., 2001; Zhao et al., 2016). This leads to aproteolytic processing of CSP, exposing its thrombospondin type-1repeat (TSR) domain, leading to an increase in parasite adhesion,which allows invasion (Coppi et al., 2005). The weak interaction ofmerozoites with erythrocytes is mediated by MSPs (Gilson et al.,2006). MSP1 is the most abundant and well-studied MSP (Cowmanand Crabb, 2006), and is considered an important target for vaccinedevelopment (Beeson et al., 2016). MSP1 is proteolyticallyprocessed by the subtilisin-like protease 1 into four fragments(denoted p42, p38, p30 and p83) (Koussis et al., 2009), which havebeen implicated in the binding to the erythrocyte surface proteinsband 3 (also known as SLC4A1) and glycophorin A (Baldwin et al.,

Box 1. Transmission and parasite biology of T. gondii,Plasmodium spp., T. cruzi, Leishmania spp. andmicrosporidiansT. gondii is transmitted through ingestion of sporozoite-containingoocysts from felids or encysted bradyzoites present in infectedundercooked meat. After ingestion, sporozoites or bradyzoites infectgastrointestinal epithelial cells in the ileum (Dubey, 2007; Dubey et al.,1997; Speer and Dubey, 1998). Bradyzoites or sporozoites thendifferentiate into tachyzoites, which spread throughout the body,generating pathology (Dubey et al., 1997; Speer and Dubey, 1998).

Infection by Plasmodium spp. occurs through the inoculation ofsporozoites into the skin of vertebrate hosts by anopheline mosquitoes(Matsuoka et al., 2002; Sidjanski and Vanderberg, 1997). Sporozoitesthen migrate to the liver and infect hepatocytes, in which they replicate(reviewed by Yang and Boddey, 2017), originating merozoites. Afterreaching the bloodstream, merozoites invade erythrocytes, in which theyare able to replicate and, thus, generate additional merozoites that canbe released and cyclically invade newerythrocytes, thereby giving rise tothe erythocytic cycle of malaria.

T. cruzi is transmitted to humans as flagellated metacyclictrypomastigotes by kissing-bug insects of the Reduviidae family(Coura and Dias, 2009; Araújo et al., 2009). These forms evolve in theinvertebrate gut from replicative forms, the epimastigotes. After bloodrepast, the vector feces containing trypomastigotes come into contactwith either the mucosa or skin micro-lesions at the bite site.Trypomastigotes can then invade any nucleated cell, where theytransform into replicative oval-shaped forms with unapparent flagella,the amastigotes, which differentiate again into trypomastigotes. Theseforms can invade neighboring cells or reach the bloodstream, therebyeither spreading to other tissues or being ingested by a new insect vector(Brener, 1973).

Leishmania spp. are transmitted by the bite of a sand fly vector, whichinoculates flagellated infective parasites, the metacyclic promastigotes,into the dermis of vertebrate hosts (Handman, 1999). Promastigotes cantransiently invade different cell types (Peters et al., 2008; Williams, 1988;Kautz-Neu et al., 2012; Moll et al., 1993; Bogdan et al., 2000;Cavalcante-Costa et al., 2019), before reaching macrophages, theirfinal destination. Inside macrophages, they nest within vacuoles, wherethey transform into replicative oval-shaped amastigote forms, causingdifferent clinical manifestations of the disease, depending on the speciesof Leishmania and on host immune responses.

Infection by microsporidians is established through the inhalation oringestion of virulent spores, which can infect several cell types (reviewedbyHan andWeiss, 2017). Upon contact with host cells, the spores extrudea long infection apparatus, thepolar tube,which drives theparasite into thehost cell. Parasites are internalized in a PV, where it replicates, generatingmore spores, which leave the host cell to infect neighboring cells, therebyamplifying infection (CDC - DPDx - Microsporidiosis; https://www.cdc.gov/dpdx/microsporidiosis/index.html).

Box2. Apicomplexans –micronemeand rhoptry secretionand gliding motility during host cell invasionMicroneme protein secretion in T. gondii and Plasmodium involves Ca2+

signaling (Dawn et al., 2014; Wetzel et al., 2004) that is initiated by itsmobilization from intracellular stores by cGMP (T. gondii) (Bullen et al.,2016) or cAMP (Plasmodium) (Dawn et al., 2014) signaling. Ca2+

increase in the cytosol activates Ca2+-dependent protein kinases,regulating microneme secretion (Lourido et al., 2010; Wetzel et al.,2004; Singh et al., 2010; Wernimont et al., 2010) as well as micronemeand PM fusion, which is mediated by the parasite protein DOC2.1 protein(Farrell et al., 2012). In T. gondii, phosphatidic acid also participates inthis secretion process by binding to the acylated pleckstrin homologydomain-containing protein (APH) that is present in micronemes (Bullenet al., 2016). Rhoptry secretion is followed by strong binding of theparasite to the host cell surface, which is mediated by specificmicroneme-secreted adhesins (Kessler et al., 2008; Singh et al.,2010). In T. gondii, MIC8 appears to participate in rhoptry secretion(Kessler et al., 2008), while in Plasmodium, the interaction of themicroneme adhesin EBA175 with its receptor glycophorin A decreasescytosolic Ca2+ levels in merozoites and triggers rhoptry secretion (Singhet al., 2010). A cytoplasmic protein homologous to the ferlin family ofCa2+-sensing proteins (TgFER2) has been shown to be essential inrhoptry secretion in T. gondii and points to a role for Ca2+ signaling in thesecretion of this organelle during invasion (Coleman et al., 2018). Inaddition, a newly identified set of proteins termed rhoptry apical surfaceproteins (RASPs) appear to be essential for rhoptry secretion in bothT. gondii and P. falciparum by promoting the rhoptry fusion with parasitePM in a Ca2+-independent manner (Suarez et al., 2019).Gliding is a substrate-dependent motility that is mediated by a protein

complex, called the glideosome (Opitz and Soldati, 2002), which has anactomyosin motor that is composed of myosin A, myosin light chain,myosin essential chain and actin filaments (Boucher and Bosch, 2015).Motility is generated after the displacement of themyosin A head from theactin filament, which occurs when the extracellular domain of atransmembrane micronemal protein (TRAP in Plasmodium and MIC2in T. gondii) strongly interacts with a receptor at the host cell surface or inthe extracellular matrix. A physical interaction between the cytoplasmictail of TRAP or MIC2 and the actin filament is mediated by a conservedprotein, named glideosome-associated connector (GAC) (Jacot et al.,2016). Thus, gliding is the result of the displacement of myosin A fromactin filaments, followed by the release of the parasite from the host cell,after the proteolytic removal of TRAP or MIC2 by an intramembraneserine protease of the rhomboid family protease (ROM4) (reviewed byFrénal et al., 2017), which then propels the parasite body forward.

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2015; Goel et al., 2003), as well as to heparin proteoglycans (Boyleet al., 2010).The key factors in the recognition and strong adhesion of

apicomplexan to the host cell surface are proteins secreted by themicronemes (and also by rhoptries in Plasmodium), the micronemeadhesive proteins (MICs). Micronemal proteins are the first to besecreted (Box 2) and are essential for both host cell adhesion andparasite motility and invasion. Secreted MICs are not resident in thePM, but the presence of hydrophobic domains on some MICsallows an individual MIC or their complexes to be embedded in theparasite membrane (Huynh et al., 2003; Meissner et al., 2002; Reisset al., 2001; Sheiner et al., 2010). MICs contain either a singleadhesive domain or a variety of combinations. Among the adhesivedomains found in T. gondiiMICs are the microneme repeat domain(MAR family) (Blumenschein et al., 2007), the type A and TSRdomains (found in the TRAP family) (Wan et al., 1997) and Apple/PAN domains (Brecht et al., 2001), as well as the chitin-binding-like(CBL) domain (Céred̀e et al., 2002). Whereas some domains, suchas TSR, are conserved among many species of Apicomplexa, othersare more restricted, such as Apple and MAR, which are specificfor coccidians (Friedrich et al., 2010). Binding assays have shownthat the MAR domain in T. gondii (Tg)MIC1 and TgMIC13selectively interacts with sialylated oligosaccharides, which arecommon terminal carbohydrates of the glycocalyx in vertebratecells (Friedrich et al., 2010). In addition, the TSR domain ofTgMIC2 is important for T. gondii motility and cell invasion(Huynh and Carruthers, 2006). Indeed, the interaction of TgMIC2type A domain with intercellular adhesion molecule 1 (ICAM-1) ispossibly involved in parasite migration across polarized epithelialcells (Barragan et al., 2005).In Plasmodium sporozoites, the micronemal proteins P36 and

P52 of 6-cysteine domain proteins (the s48/s45 family) are essential

for hepatocyte invasion (Ishino et al., 2005). P52 is a membraneGPI-anchored protein and acts as scaffold for P36 (Arredondo et al.,2018). This scaffold is then responsible for the interaction withdifferent receptors at the hepatocyte surface, such as CD81 andscavenger receptor BI (SR-BI, also known as SCARB1) forP. falciparum and P. vivax sporozoites, respectively (Manzoniet al., 2017). Regarding merozoites, here, proteins belonging to theDuffy binding-like family (DBL), reticulocyte binding-like (RBL)family and TRAP family (containing TSR and type A domains)(Boucher and Bosch, 2015; Beeson et al., 2016) are responsible forerythrocyte invasion. Microneme DBLs (only found in Plasmodiumspp.) recognize different receptors, such as sialylated glycoproteins(e.g. glycophorins) by erythrocyte-binding antigens (EBAs)(Cowman et al., 2017) or specific proteins, as for the binding ofthe DBP in P. vivax to Duffy antigen (also known as ACKR1)located on red blood cells (Adams et al., 1992; Batchelor et al.,2014). Proteins of the rhoptry RBL family in P. falciparummediatea sialic acid-independent invasion; for example, Rh4 binds tocomplement receptor 1 (CR1) and Rh5 to basigin, which are bothpresent on the erythrocyte PM (Crosnier et al., 2011; Tham et al.,2010). During invasion, P. falciparum (Pf)Rh5 forms a complexwith two other proteins, Rh5-interacting protein (PfRipr) andcysteine-rich protective antigen (CyRPA) (Chen et al., 2011; Dreyeret al., 2012). This possibly mediates the formation of a pore throughthe membranes of both merozoite and erythrocyte, which allowsthe release of Ca2+ or the injection of parasite proteins insideerythrocytes (Volz et al., 2016; Weiss et al., 2015). Owing to itsessential role in basigin binding during erythrocyte invasion bymerozoites, Rh5 is a leading candidate antigen for vaccinedevelopment against P. falciparum infection (Payne et al., 2017).Microneme TRAP family members, such as those relevant forT. gondii, also have a role in Plasmodium spp. zoite motility and areimportant for skin-to-liver migration and cell invasion (Morahanet al., 2009; Moreira et al., 2008; Sultan et al., 1997). Compared toT. gondii, TRAP members in Plasmodium spp. are more diverse,and specific TRAP proteins are expressed in the different zoitestages (Morahan et al., 2009). A recent study has shown that thehuman cell-surface integrin αvβ3 could act as a receptor for TRAPin sporozoites (Dundas et al., 2018). In merozoites, MTRAP bindsto semaphorin-7A at the erythrocyte surface (Bartholdson et al.,2012), and is important not only for motility and invasion, but alsofor gamete egress from erythrocytes inside the insect vector(Bargieri et al., 2016; Baum et al., 2006).

The formation of the moving junctionRhoptry proteins are subcompartmentalized into two distinctregions with different functions during the invasion process(Counihan et al., 2013); neck proteins act in the formation of astructure called the moving junction (MJ) or tight junction (Besteiroet al., 2011), whereas proteins from the bulb have a broad spectrumof functions, such as in cell adhesion (Beeson et al., 2016),parasitophorous vacuole (PV) formation (Ghosh et al., 2017) andimmune evasion (Hakimi et al., 2017). The existence of a MJ wasfirst described by electron microscopy observations of Plasmodiumknowlesi and is morphologically characterized by the closeapposition between the parasite and host cell PMs, associatedwith an electron-dense region right below the host cell PM (Aikawaet al., 1978) (Fig. 1A). MJs are conserved in T. gondii (Fig. 1B) andPlasmodium spp., and their main molecular components are therhoptry neck proteins (RON)2, RON4 and RON5, as well as themicroneme protein apical membrane antigen 1 (AMA1), atransmembrane protein secreted at the apical cap of the zoite

GlossaryZoite: a general name for the infective forms of Apicomplexan parasitesthat invade cells.Oocyst: infective form of T. gondii delivered in feline feces (whichcontains infective sporozoites) that can be ingested by the mammalianhost.Bradyzoite: infective and resistant form of T. gondii found in host-tissuecysts that can be ingested by the mammalian host.Tachyzoite: infective and replicative form of T. gondii responsible forparasite spread throughout different host tissues.Sporozoite: infective form of Plasmodium spp.; it is inoculated by theinsect vector in the host dermis. Sporozoites travel along host bodybloodstream and invade hepatocytes in the liver originating theexoerythrocytic phase of malaria.Merozoite: infective form of Plasmodium spp.; it is first delivered by theinfected hepatocyte, reaches the bloodstream and then invades hosterythrocytes. After replication inside erythrocytes, more merozoites arecyclically delivered, giving rise to the erythrocytic phase of malaria.Trypomastigote: infective form of T. cruzi present in the feces of theinsect vector; it is deposited in host skin during vector blood meal. Thisform is also found in the mammalian host bloodstream after parasitereplication as the amastigote form and is responsible for parasite spreadthroughout different host tissues.Amastigote: intracellular replicative form of T. cruzi or Leishmania spp.found in host cell cytosol or whiting vacuoles, respectively.Epimastigote: extracellular replicative form of T. cruzi found in theintestine of the insect vector. This form originates the trypomastigoteform within the insect vector.Promastigote: infective form of Leishmania spp.; it is inoculated by theinsect vector in host dermis during vector blood meal.

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(Alexander et al., 2005; Lebrun et al., 2005; Bradley et al., 2005;Besteiro et al., 2009; Curtidor et al., 2011; Narum et al., 2008).Additionally, RON4L1 and RON8 are found in T. gondii MJs(Guérin et al., 2017; Straub et al., 2009), and RON2 is inserted intothe host PM, acting as a receptor for AMA1, which is located on theparasite apex (Besteiro et al., 2009; Cao et al., 2009; Lamarqueet al., 2011; Richard et al., 2010). Thus, zoites provide their owninteracting ligand on the host cell to mediate the invasion process.The interaction between RON2 and AMA1 is of high affinity; thus,once the MJ is formed, zoites are committed to invasion (Delgadilloet al., 2016; Srinivasan et al., 2011), which then occurs withinseconds (Morisaki et al., 1995). Whereas RON2 and AMA1 arelocalized on the interface between the zoite and host cell surfaces,RON4, RON4L1, RON5 and RON8 are localized in the hostcytoplasm (Beck et al., 2014; Guérin et al., 2017; Straub et al.,2011). During MJ assembly, cytosolic RONs in T. gondii cooperatein recruiting host adaptor proteins, such as CIN85 (also known asSH3KBP1), CD2AP and the ESCRT-I components ALIX (alsoknown as PDCD6IP) and TSG101, which might facilitate thephysical interaction between the RON complex and the corticalactin cytoskeleton (Guérin et al., 2017) (Fig. 1B). Furthermore,secretion of toxofilin by T. gondii during invasion leads todisassembly of the actin meshwork at the site of entrance,permitting anchorage of the RON complex to a newly formed,ring-shaped F-actin structure in the MJ region (Gonzalez et al.,2009; Delorme-Walker et al., 2012). Such a ring-shaped F actinstructure was also observed at the site of MJ during Plasmodiumsporozoite invasion (Gonzalez et al., 2009). By contrast, an actinreorganization in erythrocytes linked to the MJ has not beenobserved during merozite invasion, but surprisingly, the presence ofthe cytoskeletal linker adducin was detected (Zuccala et al., 2016).The association of cytosolic RON proteins with the corticalcytoskeleton at the site of zoite entrance allows the MJ-anchoredzoite to use the traction force of its actomyosin motor to make aninvagination in the host PM (Bichet et al., 2014; Gonzalez et al.,2009; Straub et al., 2011). During T. gondii invasion, the MJselectively excludes host transmembrane proteins from the formingPVmembrane; this might prevent its fusion with the host lysosomes(Bichet et al., 2014; Charron and Sibley, 2004; Mordue et al., 1999),which would be deleterious to the parasite. Furthermore, duringT. gondii invasion, disassembly of the actin meshwork underlyingthe PM, which is caused by the secretion of profilin during the initialhost–pathogen interaction, also acts in decreasing the resistance ofhost cell membrane invagination (Delorme-Walker et al., 2012).Similarly, a cytoskeleton reorganization at erythrocyte entry site isalso required for P. falciparum invasion (Dasgupta et al., 2014).This process is regulated by Ca2+ signaling and phosphorylationof several erythrocyte cytoskeletal proteins, including β-spectrin,PIEZO1 and band 3, leading to a destabilization of the cytoskeleton(Fernandez-Pol et al., 2013; Wernimont et al., 2010; Zuccala et al.,2016). After invasion, the newly formed PV pinches off from thePM through an as-yet elusive mechanism. Experiments using thedynamin inhibitor dynasore in T. gondii suggest that the host celldynamin participates in the pinching off of the PV (Caldas et al.,2009). However, more recently, it has been shown that the pinch offof the T. gondii PV is independent of the host cell, and appears to beinduced by a twisting motion of the parasite, which mechanicallymediates membrane scission and PV detaching (Pavlou et al., 2018).

Kinetoplastids – Trypanosoma cruzi and Leishmania spp.Kinetoplastids are flagellated protozoans characterized by thepresence of a large and unique mitochondrion. Close to the

flagellar bag, this mitochondrion presents a condensation of itsDNA, named the kinetoplast, which can be easily visualized instained cells. The order Kinetoplastida is composed of several parasiteprotists of different genera that infect several animals, includinghumans. Important pathogenic species of this order are Trypanosomacruzi (the agent of Chagas disease) and different species of the genusLeishmania (agents of leishmaniasis) (Fig. 2; Box 1).

Cell invasion by Trypanosoma cruziInfection by T. cruzi starts with the binding of infectivetrypomastigote forms (see Glossary) to the host cell PM, whichinduces signaling pathways that culminate in its internalization intoan endocytic vacuole (reviewed by Andrade and Andrews, 2004;Fernandes and Andrews, 2012). Early studies of T. cruzi–host-cellinteraction demonstrated that, soon after invasion, T. cruzi resides inan acidic vacuole that contains lysosomal markers (Milder andKloetzel, 1980; de Carvalho and de Souza, 1989). However, becauseinvasion is not only independent of actin filaments (Schenkman et al.,1991), but also even facilitated by their disruption (Schenkman et al.,1991), phagocytosis can be excluded as a mechanism of parasiteentry into host cells. This corroborates the fact that T. cruzi is able toinvade any nucleated cell, including non-professional phagocyticcells (Fig. 2A,B), such as myocytes, which are actually the maintarget cells of the parasite during chronic infection in the vertebratehost (Calvet et al., 2012).

The attachment of T. cruzi provokes a rise in Ca2+ in the host cellcytosol, which signals the recruitment of host cell lysosomes tothe parasite attachment site (Rodríguez et al., 1996) (Figs 2A, 4B).Lysosomes then fusewith the host cell PM, releasing their content tothe external milieu. In doing so, they provide the membrane for thenascent PV, leading to parasite internalization. After completeenveloping of the parasite, the PV, which is rich in lysosomal-associated membrane proteins (LAMPs), is pinched off into thecytoplasm through as-yet-unidentified mechanisms. The source ofthe increased Ca2+ in the host cell cytosol is either intracellularstores, mobilized by signaling triggered by surface and secretedproteins originating from the parasite, or from the extracellularmilieu, which can influx through parasite-induced micro-injuriesinflicted on the host cell PM (Rodríguez et al., 1997; Rodríguezet al., 1999; Fernandes et al., 2011). The understanding of this entrymechanism came from studies of a basic physiological process usedby nucleated cells to repair PM wounds. Plasma membrane repair(PMR) is driven by (1) Ca2+ influx, (2) lysosomal exocytosis and(3) extensive Ca2+-dependent and actin-independent endocytosis(Bi et al., 1995; Togo et al., 2000; Reddy et al., 2001; Idone et al.,2008). During PMR, one of the enzymes secreted by the lysosomes,acid sphingomyelinase (ASM), is able to trigger endocytosisthrough the formation of ceramide at the cell surface, whichfacilitates inward budding and thus endocytosis of the woundedmembrane (McIntosh et al., 1992; Brown and London, 2000; Tamet al., 2010). Based on these findings, Fernandes and co-workersproposed that T. cruzi subverts PMR for entry into the host cell(Fernandes et al., 2011). Further evidence of PMR involvementin T. cruzi invasion comes from the observation that recentlyinternalized parasites are found in ceramide-rich vacuoles that areformed by the action of ASM during parasite-induced PMR(Fernandes et al., 2011).

Several parasite surface proteins involved in invasion have beendescribed (Ruiz et al., 1998; Caler et al., 1998; Scharfstein et al.,2000; Neira et al., 2003; Kojin et al., 2016; Alves and Colli, 2008;reviewed by Maeda et al., 2012). Among them are the members ofthe gp85/trans-sialidase (TS) super-family (gp82, gp90, gp85/TS,

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gp30); of these, gp82 has been recently shown to be recognized bythe host-cell lysosomal protein LAMP-2, which may be presentat low levels on the host-cell surface (Rodrigues et al., 2019).Binding of gp82 to target cells induces lysosome spreading andexocytosis, culminating in parasite internalization (Martins et al.,2011; Cortez et al., 2016). Two parasite enzymes have also beenimplicated in the induction of Ca2+ signaling in host cells.Oligopeptidase B appears to generate a product that is recognizedby a G-protein-coupled receptor (Tardieux et al., 1994; Burleigh andAndrews, 1995), while the cysteine protease cruzipain cleaves hostkininogen into bradykinin, which interacts with its classicalbradykinin receptor on the host (Scharfstein et al., 2000). Bothstimuli eventually converge in a pathway that involves Ca2+

signaling-mediated events and host cell lysosome recruitment,resulting in parasite internalization (Rodríguez et al., 1999; Caleret al., 1998). T. cruzi trypomastigotes can also interact with hostextracellular matrix components, which appears to facilitate theircontact with and invasion into host cells (Santana et al., 1997; Alvesand Colli, 2008; Nde et al., 2012).It is important to mention that amastigotes (see Glossary), which

can be released extracellularly are also able to invade host cells(Fernandes et al., 2013). In contrast to trypomastigotes, however,invasion of amastigotes into host cells occurs through an actinpolymerization-dependent pathway (Fernandes et al., 2013), andamastigotes are also able to induce phagocytosis in non-professional phagocytic cells (Ferreira et al., 2019).

Cell invasion by Leishmania spp.Histological tissue sections of mammalians hosts with leishmaniasisreveal that macrophages are the ultimate host cells for Leishmaniaspp. (Benchimol and de Souza, 1981; Berman et al., 1981; Brazil,1984; Davies et al., 1988). However, the circumstances under whichmacrophages are invaded in vivo by promastigotes (see Glossary)are not completely understood, particularly with regard to themolecular details. Nevertheless, in vitro, phagocytosis has beensuggested as the main route of promastigote invasion, because, inthe absence of host cell actin polymerization, macrophage infectionis impaired (Akiyama and Haight, 1971; Alexander, 1975; Ardehaliet al., 1979; Zenian et al., 1979; Roy et al., 2014) (Fig. 4C).

In vitro, phagocytosis of promastigotes by macrophages (Fig. 2D)appears to start only after two minutes of contact with parasites(Aikawa et al., 1982). It is remarkable that, during the first momentsof contact, 90% of promastigotes are attached, with low affinity,to macrophages (Uezato et al., 2005) through their flagellar tip(Aikawa et al., 1982), suggesting a role for this structure in triggeringphagocytosis. It is worth pointing out that promastigotes generallymove in the direction of the flagellum, an anterior structure (Krügerand Engstler, 2015), that has been proposed to have a role in sensingthe environment (Rotureau et al., 2009), probably as it is the firststructure to encounter a host cell. After five minutes, however,parasites are tightly bound tomacrophages and phagocytosed with nopreferred orientation (Aikawa et al., 1982; Uezato et al., 2005). Thevast majority of phagocytosed promastigotes clearly localize inside

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 parasitenuclei, and the parasite kinetoplast (blue). The red arrow indicates an extracellular T. cruzi trypomastigote, while the white arrow points to a recently internalizedparasite with lysosomes being recruited to the PV. The green arrow points to a parasite inside a PV that is covered with lysosomal markers. (B) Atrypomastigote form of T. cruzi infecting an adipocyte host-cell (HC). Image republished with permission of the American Society for Biochemistry and MolecularBiology, from Combs et al., 2005; permission conveyed through the Copyright Clearance Center. (C) Immunofluorescence microscopy image of a Leishmaniapromastigote (red, stained by anti-LPG antibody; red arrow indicates the still extracellular parasite body) invading a fibroblast by its flagellar tip through therecruitment of host cell lysosomes (green). Host cell nuclei are stained by DAPI. Adapted with permission from Cavalcante-Costa et al. (2019). (D) Scanningelectron-microscopy of a Leishmania promastigote being captured through phagocytosis by amacrophage. Thewhite arrow points to themacrophage phagocyticcup and the black arrow to the parasite body. Adapted with permission from Zenian et al. (1979).

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phagosomes (Courret et al., 2002), which promptly fuse withlysosomes (James et al., 2006; Courret et al., 2002); this creates anappropriate milieu for their fully differentiation into amastigotes (seeGlossary), and subsequent replication (Alexander and Vickerman,1975; Chang and Dwyer, 1976; Moradin and Descoteaux, 2012).Several macrophage receptors have been described to mediate

parasite adherence and subsequent phagocytosis signaling (Mosserand Rosenthal, 1993; Lefev̀re et al., 2013; Polando et al., 2018,2018; Stafford et al., 2002; reviewed by Naderer et al., 2005 andUeno and Wilson, 2012; Chauhan et al., 2017; von Stebut andTenzer, 2018). One such type of receptor, the endocytic mannosereceptors (MRs) in macrophages, which are pattern recognitionreceptors (PRRs), were previously considered to be necessary forattachment and phagocytosis of L. donovani and L. infantumpromastigotes by macrophages (Blackwell, 1985; Wilson andPearson, 1986; Chakraborty et al., 1998; Ueno et al., 2009;Polando et al., 2018). However, more recently, their roles have beencontested based on several lines of evidence. First, MR ligands canalso inhibit the uptake of L. donovani and L. major in the absence ofMRs (Akilov et al., 2007). Secondly, inhibition of parasite uptakeby MR ligands does not occur for all stages of promastigotes (Uenoet al., 2009) or all species (Da Silva et al., 1989; Mosser andHandman, 1992), and, finally, MR-depleted C57BL/6 macrophagesdo not differ in their infection rate compared to wild-type cellswith regard to various aspects of the disease outcome, suggestingthe existence of redundant infection receptors (Akilov et al., 2007).One explanation for these conflicting findings may be thatmannose-carrying ligands in promastigotes can also bind to otherC-type lectin receptors (McGreal et al., 2005) and Toll-like receptor(TLR) family members (Roeder et al., 2004). Another PRR, TLR-2,has also been implicated in the binding and internalization ofL. major by BALB/c macrophages through its interaction withlipophosphoglycan (LPG) (Srivastava et al., 2013), a highlyprevalent surface glycoconjugate in promastigotes.In addition to PRRs, it is well known that complement receptor 3

(CR3; comprising integrin αM and integrin β2) (Russell andWright, 1988), which also binds to LPG (Van Strijp et al., 1993;Talamás-Rohana et al., 1990) and fibronectin receptors (Soteriadouet al., 1992; Rizvi et al., 1988; Brittingham et al., 1999), bind togp63, a major leishmanial GPI-anchored surface metalloproteinase.LPG and gp63 have been reported to be important for internalizationof promastigotes by macrophages (Brittingham et al., 1999;Gorocica et al., 2000). However, other results, which showed thatLPG-deficient (Ilg, 2000; Späth et al., 2003) or gp63-deficient(Joshi et al., 2002) mutant promastigotes are as efficient as wild-type parasites in invading macrophages in vitro or infecting mice,indicate that these molecules are not critical for invasion, at least forthe Leishmania species studied.Although, the direct binding of promastigotes to macrophages that

occurs in vitro could also take place in vivo, the latter scenario isconsiderably more complex. Thus, it is very likely that opsonizationmay have a significant role in parasite uptake, as they most probablyinteract first with other host molecules, which would facilitatephagocytosis. In this regard, a major molecule is complementcomponent 3 (C3), which, in vitro, mainly binds to gp63 and LPGafter complement activation (Podinovskaia and Descoteaux, 2015;Sacks, 1992). The generated fragment C3b has been reported to beessential for promastigote entry intomacrophages through binding toCR1 (Da Silva et al., 1989), while iC3b, a product of C3b cleaved bygp63, opsonizes the parasites by binding to CR3 (Brittingham et al.,1995). Other opsonizing molecules, including fibronectin (Vannier-Santos et al., 1991), C-reactive protein (Bodman-Smith et al., 2002)

and heparan sulfate (Maciej-Hulme et al., 2018) have also beenimplicated in the internalization of promastigotes by macrophages.

Moreover, and importantly, even though macrophages are thefinal and main destination of parasites during the chronic phase ofleishmaniasis, and may actually be directly infected bypromastigotes in vivo (Peters et al., 2008), neutrophils are themajor cells to capture promastigote during the initial phase ofinfection (Peters et al., 2008). In this case, macrophages may alsobecome secondarily infected by ingesting Leishmania-containingapoptotic bodies after neutrophil death. This appears to be crucialfor the survival of parasites and the outcome of infection (Afonsoet al., 2008) and has been earlier referred to as the ‘Trojan horsestrategy’ (Laskay et al., 2003).

Dendritic cells (DCs) are also targets for Leishmania spp.invasion. However, invasion of DCs is reported to occur mostly atlater phases of the disease when susceptible promastigotes hadeither died through complement-mediated lysis or transformed intoamastigotes inside macrophages, and when antibodies to parasitesappear in sera. In fact, it has been shown that DCs preferentially takeup amastigotes, rather than promastigotes, requiring opsonizationthrough their receptor FcγR, which recognizes IgG (Guy andBelosevic, 1993; Peters et al., 1995; Kima et al., 2000).

Much less is known about the direct invasion of amastigotes ortheir transfer from one host cell to another, although this has beenreported to occur via macrophage receptors for Fcγ orphosphatidylserine (Love et al., 1998; Kane and Mosser, 2000;De Freitas Balanco et al., 2001; Wanderley et al., 2006),phagocytosis of infected apoptotic bodies (Peters et al., 2008) orby direct cell-to-cell transfer of PV extrusions (Real et al., 2014).

Although the inhibition of phagocytosis mostly blocks invasionof macrophages by promastigotes, a small number of parasites canstill be found inside these cells (Roy et al., 2014; Lewis, 1974;Aikawa et al., 1982), indicating that Leishmania can invademacrophages by routes other than classical phagocytosis. In fact,it has long been described that promastigotes can also invade non-phagocytic cells (Bogdan et al., 2000;Minero et al., 2004; Holbrookand Palczuk, 1975; Schwartzman and Pearson, 1985). Recent workfrom our group has unveiled a new non-phagocytic route ofpromastigote invasion without any involvement of the host cellcytoskeleton, thereby providing definitive evidence for an entrymechanism other than phagocytosis (Cavalcante-Costa et al., 2019).This pathway involves the Ca2+-dependent recruitment andexocytosis of host cell lysosomes to the parasite invasion site,where they instantly fuse with the PM, much like in the invasionmechanism described above for T. cruzi. This creates a niche for theinternalization of promastigotes, which most often occurs throughthe flagella (Cavalcante-Costa et al., 2019) (Fig. 2C). It is possible,therefore, to speculate that, in vivo, Leishmania does not solely relyon being captured by phagocytes to establish infection, and thatdifferent infected cell types of the dermis, as already reported byother groups (Locksley et al., 1988; Bogdan et al., 2000), could actas Trojan horses.

MicrosporidiansMicrosporidians have long been considered as primitive protozoanswith which they share some morphological similarities, but moreaccurate phylogenetic analysis have shown that they are, in fact,related to fungi (Weiss et al., 1999; James et al., 2006). Theseparasites were described by Louis Pasteur as a plague that decimatedsilkworms with a huge impact in silk industry in France, almost150 years ago (Pasteur and Pasteur, 1870). Microsporidians arespread through parasite spores, which can infect not only several

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animals of economic importance, but also humans, being importantpathogens. The phylum Microsporidia is composed by over 140genera, but only eight have been characterized as causative ofhuman microsporidiosis: Enterocytozoon, Encephalytozoon,Pleistophora, Trachipleistophora, Vittaforma, Brachiola, Septataand Nosema (Weiss, 2000). In humans, these parasites are able toinfect almost every organ system causing asymptomatic, chronic orlethal infections, depending on the immunological status of thepatient (Weber et al., 2000, 1994) (Box 1).

Cell invasion by microsporidiansMicrosporidian spores are rigid and round, and range from 1 to12 µm in size, depending on the species; they can be divided in threebasic morphological structures: the spore, the sporoplasm, whichis the parasite body, and the invasion apparatus (Han and Weiss,2017). The spore wall is mainly composed of chitin andglycoproteins (Han and Weiss, 2017). The most important familyof proteins that form the spore wall are named spore wall proteins(SWPs), and these are essential for the formation of the spore butalso participate in the adhesion of the spore to host cell PM beforethe formation of the invading apparatus, named the polar tube(recently reviewed by Yang et al., 2018) (Figs 3, 4D). The polar tubeis a coiled structure, which is extruded in a fraction of a second, as aresult of osmotic changes inside the spore (Fig. 3A,B). It can be aslong as 500 µm, depending on the species, with a narrow diameterof between 0.1 and 0.2 µm, through which the sporoplasm travels toinfect the host cell (Weidner, 1976; Han and Weiss, 2017). Polartube proteins (PTPs), which form the polar tube, are the main factorsinvolved in invasion. At least five PTPs have been described inmicrosporidians, with PTP1, PTP2, PTP3 and PTP5 found evenlydistributed throughout the polar tube, whereas PTP4 is specificallylocated to the polar tube tip, in close interaction with host cell PM atthe infection synapsis (Han et al., 2017). PTP4 binds to transferrinreceptor 1 (TfR1, also known as TFRC) on the PM of mammaliancells, and blocking this binding with specific antibodies has been

shown to reduce infections in vitro, making this protein a promisingnew target for drug and vaccine development (Han et al., 2017;reviewed by Han and Weiss, 2017).

Even thoughmicrosporidians have been known about and studiedfor more than a century, essential features, such as the mechanism ofdirect injection into the cytoplasm and the endocytosis of theinfective sporoplasm by the host cell still remain a matter ofdiscussion. Interestingly, it has been shown that if the spore isphagocytosed by macrophages, the extrusion of the polar tube,typically termed germination, occurs from within the PV, leading tothe perforation of the vacuolar membrane and the delivery of theinfectious sporoplasm directly into the cytosol (Franzen, 2005); thisconstitutes a means to evade the deleterious conditions withinfusogenic vacuoles. It is also interesting to note that microsporidiansare able to replicate inside PVs, which are modified by the parasiteto escape lysosomal acidification and to allow nutrient uptake fromthe cytoplasm (Rönnebäumer et al., 2008). Indeed, the membrane ofthe PV that contains Encephalitozoon cuniculi has been shown tooriginate from the host cell PM (Fig. 3C), as demonstrated by theaddition of dyes to the host cell PM, which were found in the PVmembrane shortly after cell invasion (Rönnebäumer et al., 2008).As PTP4 localizes to the tip of the polar tube and is able to interactwith the host cell PM, Han et al. have proposed a model, in whichPTP4 together with PTP1 helps forming the invagination of the hostcell PM that excludes the extracellular environment, thus creatinga microenvironment that is protected from host innate immuneresponses, thereby facilitating invasion (Han et al., 2017). However,the machinery for pinching off and fission, which subsequentlydetaches the nascent membrane of the PV is still unknown. It hasbeen hypothesized that invasion could also involve the clathrinmediated-endocytosis machinery (Han et al., 2017), or host cellactin polymerization (Foucault and Drancourt, 2000). Althoughthese obligatory intracellular parasites have the smallest genomeamong eukaryotes, they have evolved one of the most remarkablemachinery to invade host cells. In this context, a better

Fig. 3. Microsporidians invade cells through the formation ofa polar tube. (A,B) Correlative light and electron microscopy(CLEM) analysis of Encephalitozoon hellem infection. Thefluorescence image and scanning electron microscopy (SEM)image of the same site were taken sequentially. Thefluorescence 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 ofreleased sporoplasm (SP). (C) Transmission electronmicroscopy (TEM) data demonstrating that the polar tube issurrounded with host cell membrane (black arrows) at the site ofinfection. 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 aCC BY 4.0 license.

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understanding of the proteins involved in spore adhesion to host cellPM (SWPs) and the ones involved in the functioning of the polartube (PTPs), as well as the discovery of the molecular actorsinvolved in PV pinch-off are crucial to unveil parasite entry process.

Concluding remarksEven though the parasites discussed here have been known andstudied for decades, some key issues of their biology and the detailsof the mechanism by which they invade host cells remain elusive.Many questions still remain open with regard to the invasionprocesses presented here. For instance, how exactly do nascent PVspinch off from the PM and which membrane fission machinery isresponsible for this crucial invasion step? Is membrane fissiondriven by the parasite itself, or does it require the recruitment ofhost cell molecules? Some attempts to solve this question may bestill controversial. For instance, results drawn from inhibitionexperiments with dynasore might be ambiguous in inferring thatdynamin participates in this process owing to the cytotoxic and off-target effects of this drug (Preta et al., 2015). Therefore, moreadvanced approaches, such as the use of CRISPR/Cas9 or RNAisilencing to target candidates, such as dynamin itself, as well asother host cell proteins involved in membrane fission are anticipatedto provide more reliable results. Importantly, as invasion does not

occur without the initial adhesion of the parasite to the host cell, thediscovery of the underlying molecules is also crucial. Another pointthat merits additional discussion is how infective Leishmaniapromastigotes that are delivered by sand flies actually reachmacrophages. We know today, for instance, that after infection ofmice with promastigotes, macrophages are not necessarily the firstcells that are invaded by the parasite (Peters et al., 2008), and that theindirect infection of macrophages through phagocytosis ofamastigotes, expelled from infected cells from PVs or as apoptoticbodies, or other mechanisms, are more likely to occur. Moreover,most conditions of in vitro infections, which are used to study thecell invasion process, do not recapitulate the in vivo situation, forinstance, the use of fresh serum as a source of complement, becauseC3 opsonization of promastigotes, which is likely to occur in vivo,strongly increases their ability to infect macrophages. Hence,findings based solely on in vitro infection of macrophages bypromastigotes should be interpreted with caution. Thus, morephysiologically relevant approaches, such as in situ visualization ofmice infections by intravital microscopy, using labeled cells andmolecules, are needed to reach a better understanding of theinvasion processes by these intracellular parasites. Similarly,experiments aiming to avoid off-target drug effects and to moreclosely reflect the processes occurring in nature would certainly be

Ca2+

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A Toxoplasma gondii Plasmodium spp.

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Moving junction-mediated entry

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Fig. 4. Overview over the cell invasionmechanisms employed by apicomplexans,kinetoplastides and microsporidians to entryhost cells. (A) (1) Apicomplexans (e.g. T. gondii andPlasmodium spp.) first weakly interact with the hostcell PM using their resident-GPI anchored proteins.At the same time, the strong interaction provided bymicroneme-secreted protein (MICs) leads to zoitere-orientation. (2) With the apical complex facing thehost cell PM, proteins secreted by the rhoptriesinteract with host cell proteins and (3) form themoving junction (see Fig. 1), which provides theforce that drives parasites into the host cellcytoplasm. Inside host cells, parasites reside in anon-fusogenic vacuole and so avoid lysosomalfusion. (B) T. cruzi invades non-phagocytic host cellsby inducing both (1) extracellular Ca2+ influx throughparasite-induced PM wounds and the release ofintracellularly stored Ca2+. (2) This leads to therecruitment of host cell lysosomes that function asCa2+-sensitive exocytic vesicles, which donatemembranes to the nascent PV. (3) Inside host cells,the vacuole keeps fusing with host cell lysosomes,leading to (4) parasites escaping into the host cellcytosol where they replicate as round-shapedamastigotes. (C) Leishmania spp. are captured byphagocytes through either (1) actin-mediatedphagocytosis or (2) by non-phagocytic cells throughCa2+-dependent recruitment of host cell lysosomesthat donate membranes to the nascent PV. Thisprocess appears to be the same as infection byT. cruzi trypromastigotes, indicating that theunderlying mechanisms are conserved in thesekinetoplastides. After invasion, Leishmania spp. liveand replicate inside acidic vacuoles that fuse withhost cell lysosomes. (D) Microsporidian spores bindto host cells with their spore wall and extrude theirpolar tube, which creates an invagination in the hostcell PM. The infective sporoplasm that harbors thespore travels along the polar tube lumen and isdelivered at the infection site. After internalization,the parasite lives and replicates inside non-fusogenic vacuoles and so avoids lysosomal fusion.

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helpful in making appropriate choices for molecular targets used indrug discovery and vaccine development.Finally, some features of the invasion processes are common

among phylogenetically-related parasites. For example, cellinvasion by Plasmodium spp. and Toxoplasma gondii zoites maydiffer in terms of the specific host cell invaded, initial adhesionmolecules or proteins used for the assembly of the MJ, but,ultimately, the same invading machinery is formed. Similarly, wenow know that the infective stages of both T. cruzi and Leishmaniaare able to induce cell invasion by subverting PMR-inducedendocytosis (Fig. 4). Thus, building on established knowledge,extrapolation of models and mechanisms from one species couldhelp to narrow knowledge gaps in closely related microorganisms,thereby accelerating new discoveries and guiding the proposal ofnew approaches to better understand the biology of these parasitesand the diseases they cause.

AcknowledgementsT. C.-G. and É. S. M.-D. would like to thank Professors Égler Chiari and Lúcia MariaC. Galva ̃o from Universidade Federal de Minas Gerais, Brazil, for all their support toour research groups and dedicate this review to their important accomplishments inthe field of protozoology, notably for their studies with Trypanosoma cruzi. We alsoacknowledge the Departamento de Parasitologia, Universidade Federal de MinasGerais, for all its support during the establishment of our research groups andlaboratories. Thiago Castro-Gomes also acknowledges Professor Norma WindsorAndrews from the University of Maryland, USA, who first described the lysosome-dependent mechanism of cell entry in Trypanosoma cruzi, for all her support andshared knowledge.

Competing interestsThe authors declare no competing or financial interests.

FundingOur work in this field is supported by FAPEMIG (Fundaça ̃o de Amparo à Pesquisado Estado de Minas Gerais), CAPES (Coordenação de Aperfeiçoamento dePessoal de Nıv́el Superior) and CNPq (Conselho Nacional de DesenvolvimentoCientıf́ico e Tecnológico).

ReferencesAdams, J. H., Sim, B. K., Dolan, S. A., Fang, X., Kaslow, D. C. and Miller, L. H.(1992). A family of erythrocyte binding proteins of malaria parasites. Proc. Natl.Acad. Sci. USA 89, 7085-7089. doi:10.1073/pnas.89.15.7085

Adl, S. M., Bass, D., Lane, C. E., Lukeš, J., Schoch, C. L., Smirnov, A., Agatha,S., Berney, C., Brown, M. W., Burki, F. et al. (2018). Revisions to theclassification, nomenclature, and diversity of eukaryotes. J. Eukaryot. Microbiol.66, jeu.12691. doi:10.1111/jeu.12691

Afonso, L., Borges, V. M., Cruz, H., Ribeiro-Gomes, F. L., DosReis, G. A., Dutra,A. N., Clarêncio, J., de Oliveira, C. I., Barral, A., Barral-Netto, M. et al. (2008).Interactions with apoptotic but not with necrotic neutrophils increase parasiteburden in human macrophages infected with Leishmania amazonensis.J. Leukoc. Biol. 84, 389-396. doi:10.1189/jlb.0108018

Aikawa, M., Miller, L. H., Johnson, J. and Rabbege, J. (1978). Erythrocyte entryby malarial parasites. A moving junction between erythrocyte and parasite. J. CellBiol. 77, 72-82. doi:10.1083/jcb.77.1.72

Aikawa, M., Hendricks, L. D., Ito, Y. and Jagusiak, M. (1982). Interactionsbetween macrophagelike cells and Leishmania braziliensis in vitro. Am. J. Pathol.108, 50-59.

Akilov, O. E., Kasuboski, R. E., Carter, C. R. andMcDowell, M. A. (2007). The roleof mannose receptor during experimental leishmaniasis. J. Leukoc. Biol. 81,1188-1196. doi:10.1189/jlb.0706439

Akiyama, H. J. and Haight, R. D. (1971). Interaction of Leishmania donovani andhamster peritoneal macrophages: a phase-contrast microscopical study.Am. J. Trop. Med. Hyg. 20, 539-545. doi:10.4269/ajtmh.1971.20.539

Alexander, J. (1975).Effect of theantiphagocytic agent cytochalasinBonmacrophageinvasion by Leishmania mexicana promastigotes and Trypanosoma cruziepimastigotes. J. Protozool. 22, 237-240. doi:10.1111/j.1550-7408.1975.tb05858.x

Alexander, J. and Vickerman, K. (1975). Fusion of host cell secondary lysosomeswith the parasitophorous vacuoles of Leishmania mexicana-infectedmacrophages. J. Protozool. 22, 502-508. doi:10.1111/j.1550-7408.1975.tb05219.x

Alexander, D. L., Mital, J., Ward, G. E., Bradley, P. and Boothroyd, J. C. (2005).Identification of the moving junction complex of Toxoplasma gondii: acollaboration between distinct secretory organelles. PLoS Pathog. 1, e17.doi:10.1371/journal.ppat.0010017

Alves, M. J. M. and Colli, W. (2008). Role of the gp85/trans-sialidase superfamily ofglycoproteins in the interaction of trypanosoma cruzi with host structures. Subcell.Biochem. 47, 58-69. doi:10.1007/978-0-387-78267-6_4

Andrade, L. O. and Andrews, N. W. (2004). Lysosomal fusion is essential for theretention of Trypanosoma cruzi inside host cells. J. Exp. Med. 200, 1135-1143.doi:10.1084/jem.20041408

Araújo, C. A. C., Waniek, P. J. and Jansen, A. M. (2009). An overview of Chagasdisease and the role of triatomines on its distribution in Brazil. Vector BorneZoonotic Dis. 9, 227-234. doi:10.1089/vbz.2008.0185

Ardehali, S. M., Khoubyar, K. and Rezai, H. R. (1979). Studies on the effect of theanti-phagocytic agent cytochalasin B on Leishmania-macrophage interaction.Acta Trop. 36, 15-21.

Arredondo, S. A., Swearingen, K. E., Martinson, T., Steel, R., Dankwa, D. A.,Harupa, A., Camargo, N., Betz, W., Vigdorovich, V., Oliver, B. G. et al. (2018).The micronemal plasmodium proteins P36 and P52 act in concert to establish thereplication-permissive compartment within infected hepatocytes. Front. Cell.Infect. Microbiol. 8, 413. doi:10.3389/fcimb.2018.00413

Azzouz, N., Kamena, F., Laurino, P., Kikkeri, R., Mercier, C., Cesbron-Delauw,M.-F., Dubremetz, J.-F., De Cola, L. and Seeberger, P. H. (2013). Toxoplasmagondii secretory proteins bind to sulfated heparin structures. Glycobiology 23,106-120. doi:10.1093/glycob/cws134

Baldwin, M. R., Li, X., Hanada, T., Liu, S. C. and Chishti, A. H. (2015). Merozoitesurface protein 1 recognition of host glycophorin a mediates malaria parasiteinvasion of red blood cells. Blood 125, 2704-2711. doi:10.1182/blood-2014-11-611707

Bargieri, D. Y., Thiberge, S., Tay, C. L., Carey, A. F., Rantz, A., Hischen, F.,Lorthiois, A., Straschil, U., Singh, P., Singh, S. et al. (2016). PlasmodiumMerozoite TRAP Family Protein Is Essential for Vacuole Membrane Disruptionand Gamete Egress from Erythrocytes. Cell Host Microbe 20, 618-630. doi:10.1016/j.chom.2016.10.015

Barragan, A., Brossier, F. and Sibley, L. D. (2005). Transepithelial migration ofToxoplasma gondii involves an interaction of intercellular adhesion molecule 1(ICAM-1) with the parasite adhesinMIC2.Cell. Microbiol. 7, 561-568. doi:10.1111/j.1462-5822.2005.00486.x

Bartholdson, S. J., Bustamante, L. Y., Crosnier, C., Johnson, S., Lea, S.,Rayner, J. C. andWright, G. J. (2012). Semaphorin-7A is an erythrocyte receptorfor P. falciparum merozoite-specific TRAP homolog, MTRAP. PLoS Pathog. 8.doi:10.1371/journal.ppat.1003031

Batchelor, J. D., Malpede, B. M., Omattage, N. S., DeKoster, G. T., Henzler-Wildman, K. A. and Tolia, N. H. (2014). Red blood cell invasion by plasmodiumvivax: structural basis for DBP engagement of DARC. PLoS Pathog. 10,e1003869. doi:10.1371/journal.ppat.1003869

Baum, J., Richard, D., Healer, J., Rug, M., Krnajski, Z., Gilberger, T. W., Green,J. L., Holder, A. A. and Cowman, A. F. (2006). A conserved molecular motordrives cell invasion and gliding motility across malaria life cycle stages and otherapicomplexan parasites. J. Biol. Chem. 281, 5197-5208. doi:10.1074/jbc.M509807200

Beck, J. R., Chen, A. L., Kim, E. W. and Bradley, P. J. (2014). RON5 is critical fororganization and function of the Toxoplasma moving junction complex. PLoSPathog. 10, e1004025. doi:10.1371/journal.ppat.1004025

Beeson, J. G., Drew, D. R., Boyle, M. J., Feng, G., Fowkes, F. J. I. and Richards,J. S. (2016). Merozoite surface proteins in red blood cell invasion, immunity andvaccines against malaria. FEMS Microbiol. Rev. 40, 343-372. doi:10.1093/femsre/fuw001

Benchimol, M. and de Souza, W. (1981). Leishmania mexicana amazonensis:attachment to the membrane of the phagocytic vacuole of macrophages in vivo.Z. Parasitenkd. 66, 25-29. doi:10.1007/BF00941942

Berman, J. D., Fioretti, T. B. and Dwyer, D. M. (1981). In Vivo and In Vitrolocalization of leishmania within macrophage phagolysosomes: use of colloidalgold as a lysosomal label. J. Protozool. 28, 239-242. doi:10.1111/j.1550-7408.1981.tb02839.x

Besteiro, S., Michelin, A., Poncet, J., Dubremetz, J.-F. and Lebrun, M. (2009).Export of a toxoplasma gondii rhoptry neck protein complex at the host cellmembrane to form the moving junction during invasion. PLoS Pathog. 5,e1000309. doi:10.1371/journal.ppat.1000309

Besteiro, S., Dubremetz, J.-F. and Lebrun, M. (2011). The moving junction ofapicomplexan parasites: a key structure for invasion. Cell. Microbiol. 13, 797-805.doi:10.1111/j.1462-5822.2011.01597.x

Bi, G. Q., Alderton, J. M. and Steinhardt, R. A. (1995). Calcium-regulatedexocytosis is required for cell membrane resealing. J. Cell Biol. 131, 1747-1758.doi:10.1083/jcb.131.6.1747

Bichet, M., Joly, C., Henni, A. H., Guilbert, T., Xémard, M., Tafani, V., Lagal, V.,Charras, G. and Tardieux, I. (2014). The toxoplasma-host cell junction isanchored to the cell cortex to sustain parasite invasive force. BMC Biol. 12, 773.doi:10.1186/s12915-014-0108-y

Blackwell, J. M. (1985). Receptors and recognition mechanisms of Leishmaniaspecies. Trans. R. Soc. Trop. Med. Hyg. 79, 606-612. doi:10.1016/0035-9203(85)90166-X

Blumenschein, T. M. A., Friedrich, N., Childs, R. A., Saouros, S., Carpenter,E. P., Campanero-Rhodes, M. A., Simpson, P., Chai, W., Koutroukides, T.,

10


Journal

ofCe

llScience

https://doi.org/10.1073/pnas.89.15.7085https://doi.org/10.1073/pnas.89.15.7085https://doi.org/10.1073/pnas.89.15.7085https://doi.org/10.1111/jeu.12691https://doi.org/10.1111/jeu.12691https://doi.org/10.1111/jeu.12691https://doi.org/10.1111/jeu.12691https://doi.org/10.1189/jlb.0108018https://doi.org/10.1189/jlb.0108018https://doi.org/10.1189/jlb.0108018https://doi.org/10.1189/jlb.0108018https://doi.org/10.1189/jlb.0108018https://doi.org/10.1083/jcb.77.1.72https://doi.org/10.1083/jcb.77.1.72https://doi.org/10.1083/jcb.77.1.72https://doi.org/10.1189/jlb.0706439https://doi.org/10.1189/jlb.0706439https://doi.org/10.1189/jlb.0706439https://doi.org/10.4269/ajtmh.1971.20.539https://doi.org/10.4269/ajtmh.1971.20.539https://doi.org/10.4269/ajtmh.1971.20.539https://doi.org/10.1111/j.1550-7408.1975.tb05858.xhttps://doi.org/10.1111/j.1550-7408.1975.tb05858.xhttps://doi.org/10.1111/j.1550-7408.1975.tb05858.xhttps://doi.org/10.1111/j.1550-7408.1975.tb05219.xhttps://doi.org/10.1111/j.1550-7408.1975.tb05219.xhttps://doi.org/10.1111/j.1550-7408.1975.tb05219.xhttps://doi.org/10.1371/journal.ppat.0010017https://doi.org/10.1371/journal.ppat.0010017https://doi.org/10.1371/journal.ppat.0010017https://doi.org/10.1371/journal.ppat.0010017https://doi.org/10.1007/978-0-387-78267-6_4https://doi.org/10.1007/978-0-387-78267-6_4https://doi.org/10.1007/978-0-387-78267-6_4https://doi.org/10.1084/jem.20041408https://doi.org/10.1084/jem.20041408https://doi.org/10.1084/jem.20041408https://doi.org/10.1089/vbz.2008.0185https://doi.org/10.1089/vbz.2008.0185https://doi.org/10.1089/vbz.2008.0185https://doi.org/10.3389/fcimb.2018.00413https://doi.org/10.3389/fcimb.2018.00413https://doi.org/10.3389/fcimb.2018.00413https://doi.org/10.3389/fcimb.2018.00413https://doi.org/10.3389/fcimb.2018.00413https://doi.org/10.1093/glycob/cws134https://doi.org/10.1093/glycob/cws134https://doi.org/10.1093/glycob/cws134https://doi.org/10.1093/glycob/cws134https://doi.org/10.1182/blood-2014-11-611707https://doi.org/10.1182/blood-2014-11-611707https://doi.org/10.1182/blood-2014-11-611707https://doi.org/10.1182/blood-2014-11-611707https://doi.org/10.1016/j.chom.2016.10.015https://doi.org/10.1016/j.chom.2016.10.015https://doi.org/10.1016/j.chom.2016.10.015https://doi.org/10.1016/j.chom.2016.10.015https://doi.org/10.1016/j.chom.2016.10.015https://doi.org/10.1111/j.1462-5822.2005.00486.xhttps://doi.org/10.1111/j.1462-5822.2005.00486.xhttps://doi.org/10.1111/j.1462-5822.2005.00486.xhttps://doi.org/10.1111/j.1462-5822.2005.00486.xhttps://doi.org/10.1371/journal.ppat.1003031https://doi.org/10.1371/journal.ppat.1003031https://doi.org/10.1371/journal.ppat.1003031https://doi.org/10.1371/journal.ppat.1003031https://doi.org/10.1371/journal.ppat.1003869https://doi.org/10.1371/journal.ppat.1003869https://doi.org/10.1371/journal.ppat.1003869https://doi.org/10.1371/journal.ppat.1003869https://doi.org/10.1074/jbc.M509807200https://doi.org/10.1074/jbc.M509807200https://doi.org/10.1074/jbc.M509807200https://doi.org/10.1074/jbc.M509807200https://doi.org/10.1074/jbc.M509807200https://doi.org/10.1371/journal.ppat.1004025https://doi.org/10.1371/journal.ppat.1004025https://doi.org/10.1371/journal.ppat.1004025https://doi.org/10.1093/femsre/fuw001https://doi.org/10.1093/femsre/fuw001https://doi.org/10.1093/femsre/fuw001https://doi.org/10.1093/femsre/fuw001https://doi.org/10.1007/BF00941942https://doi.org/10.1007/BF00941942https://doi.org/10.1007/BF00941942https://doi.org/10.1111/j.1550-7408.1981.tb02839.xhttps://doi.org/10.1111/j.1550-7408.1981.tb02839.xhttps://doi.org/10.1111/j.1550-7408.1981.tb02839.xhttps://doi.org/10.1111/j.1550-7408.1981.tb02839.xhttps://doi.org/10.1371/journal.ppat.1000309https://doi.org/10.1371/journal.ppat.1000309https://doi.org/10.1371/journal.ppat.1000309https://doi.org/10.1371/journal.ppat.1000309https://doi.org/10.1111/j.1462-5822.2011.01597.xhttps://doi.org/10.1111/j.1462-5822.2011.01597.xhttps://doi.org/10.1111/j.1462-5822.2011.01597.xhttps://doi.org/10.1083/jcb.131.6.1747https://doi.org/10.1083/jcb.131.6.1747https://doi.org/10.1083/jcb.131.6.1747https://doi.org/10.1186/s12915-014-0108-yhttps://doi.org/10.1186/s12915-014-0108-yhttps://doi.org/10.1186/s12915-014-0108-yhttps://doi.org/10.1186/s12915-014-0108-yhttps://doi.org/10.1016/0035-9203(85)90166-Xhttps://doi.org/10.1016/0035-9203(85)90166-Xhttps://doi.org/10.1016/0035-9203(85)90166-Xhttps://doi.org/10.1038/sj.emboj.7601704https://doi.org/10.1038/sj.emboj.7601704

Blackman, M. J. et al. (2007). Atomic resolution insight into host cell recognitionby Toxoplasma gondii. EMBO J. 26, 2808-2820. doi:10.1038/sj.emboj.7601704

Bodman-Smith, K. B., Mbuchi, M., Culley, F. J., Bates, P. A. and Raynes, J. G.(2002). C-reactive protein-mediated phagocytosis of Leishmania donovanipromastigotes does not alter parasite survival or macrophage responses.Parasite Immunol. 24, 447-454. doi:10.1046/j.1365-3024.2002.00486.x

Bogdan, C., Donhauser, N., Döring, R., Röllinghoff, M., Diefenbach, A. andRittig, M. G. (2000). Fibroblasts as host cells in latent leishmaniosis. J. Exp. Med.191, 2121-2130. doi:10.1084/jem.191.12.2121

Boucher, L. E. andBosch, J. (2015). The apicomplexanglideosomeandadhesins–Structures and function. J. Struct. Biol. 190, 93-114. doi:10.1016/j.jsb.2015.02.008

Boyle, M. J., Richards, J. S., Gilson, P. R., Chai, W. and Beeson, J. G. (2010).Interactions with heparin-like molecules during erythrocyte invasion byPlasmodium falciparum merozoites. Blood 115, 4559-4568. doi:10.1182/blood-2009-09-243725

Bradley, P. J.,Ward, C., Cheng, S. J., Alexander, D. L., Coller, S., Coombs, G. H.,Dunn, J. D., Ferguson, D. J., Sanderson, S. J., Wastling, J. M. et al. (2005).Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii. J. Biol. Chem. 280, 34245-34258.doi:10.1074/jbc.M504158200

Brazil, R. P. (1984). In vivo fusion of lysosomes with parasitophorous vacuoles ofLeishmania-infected macrophages. Ann. Trop. Med. Parasitol. 78, 87-91. doi:10.1080/00034983.1984.11811781

Brecht, S., Carruthers, V. B., Ferguson, D. J. P., Giddings, O. K., Wang, G.,Jäkle, U., Harper, J. M., Sibley, L. D. and Soldati, D. (2001). The toxoplasmamicronemal protein MIC4 is an adhesin composed of six conserved appledomains. J. Biol. Chem. 276, 4119-4127. doi:10.1074/jbc.M008294200

Brener, Z. (1973). Biology of trypanosoma cruzi. Annu. Rev. Microbiol. 27, 347-382.doi:10.1146/annurev.mi.27.100173.002023

Brittingham, A., Morrison, C. J., McMaster, W. R., McGwire, B. S., Chang, K. P.and Mosser, D. M. (1995). Role of the Leishmania surface protease gp63 incomplement fixation, cell adhesion, and resistance to complement-mediated lysis.J. Immunol. 155, 3102-3111. doi:10.1016/0169-4758(95)80054-9

Brittingham, A., Chen, G., McGwire, B. S., Chang, K.-P. and Mosser, D. M.(1999). Interaction of Leishmania gp63 with cellular receptors for fibronectin.Infect. Immun. 67, 4477-4484. doi:10.1128/IAI.67.9.4477-4484.1999

Brown, D. A. and London, E. (2000). Structure and function of sphingolipid- andcholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221-17224. doi:10.1074/jbc.R000005200

Bullen, H. E., Jia, Y., Yamaryo-Botté, Y., Bisio, H., Zhang, O., Jemelin, N. K.,Marq, J.-B., Carruthers, V., Botté, C. Y. and Soldati-Favre, D. (2016).Phosphatidic acid-mediated signaling regulates microneme secretion intoxoplasma. Cell Host Microbe 19, 349-360. doi:10.1016/j.chom.2016.02.006

Burleigh, B. A. and Andrews, N. W. (1995). A 120-kDa Alkaline Peptidase fromTrypanosoma cruzi Is Involved in the Generation of a Novel Ca2+ -signaling Factorfor Mammalian Cells. J. Biol. Chem. 270, 5172-5180. doi:10.1074/jbc.270.10.5172

Caldas, L. A., Attias, M. and de Souza, W. (2009). Dynamin inhibitor impairsToxoplasma gondii invasion. FEMS Microbiol. Lett. 301, 103-108. doi:10.1111/j.1574-6968.2009.01799.x

Caler, E. V., Vaena De Avalos, S., Haynes, P. A., Andrews, N. W. and Burleigh,B. A. (1998). Oligopeptidase B-dependent signaling mediates host cell invasionby Trypanosoma cruzi. EMBO J. 17, 4975-4986. doi:10.1093/emboj/17.17.4975

Calvet, C. M., Melo, T. G., Garzoni, L. R., Oliveira, F. O. R., Silva Neto, D. T.,Meirelles, M. N. S. L. and Pereira, M. C. S. (2012). Current understanding of theTrypanosoma cruzi-cardiomyocyte interaction. Front. Immunol. 3. doi:10.3389/fimmu.2012.00327

Cao, J., Kaneko, O., Thongkukiatkul, A., Tachibana, M., Otsuki, H., Gao, Q.,Tsuboi, T. and Torii, M. (2009). Rhoptry neck protein RON2 forms a complex withmicroneme protein AMA1 in Plasmodium falciparummerozoites.Parasitol. Int. 58,29-35. doi:10.1016/j.parint.2008.09.005

Carruthers, V. and Boothroyd, J. C. (2007). Pulling together: an integrated modelof Toxoplasma cell invasion. Curr. Opin. Microbiol. 10, 83-89. doi:10.1016/j.mib.2006.06.017

Carruthers, V. B. and Sibley, L. D. (1997). Sequential protein secretion from threedistinct organelles of Toxoplasma gondii accompanies invasion of humanfibroblasts. Eur. J. Cell Biol. 73, 114-123.

Cavalcante-Costa, V. S., Costa-Reginaldo, M., Queiroz-Oliveira, T., Oliveira,A. C. S., Couto, N. F., DosAnjos, D. O., Lima-Santos, J., Andrade, L. O., Horta,M. F. and Castro-Gomes, T. (2019). Leishmania amazonensis hijacks host celllysosomes involved in plasma membrane repair to induce invasion in fibroblasts.J. Cell Sci. 132, jcs226183. doi:10.1242/jcs.226183

Céred̀e, O., Dubremetz, J. F., Bout, D. and Lebrun, M. (2002). The Toxoplasmagondii protein MIC3 requires pro-peptide cleavage and dimerization to function asadhesin. EMBO J. 21, 2526-2536. doi:10.1093/emboj/21.11.2526

Chakraborty, R., Chakraborty, P. and Basu, M. K. (1998). Macrophage mannosylfucosyl receptor: Its role in invasion of virulent and avirulent L. donovanipromastigotes. Biosci. Rep. 18, 129-142. doi:10.1023/a:1020192512001

Chang, K. and Dwyer, D. (1976). Multiplication of a human parasite (Leishmaniadonovani) in phagolysosomes of hamster macrophages in vitro. Science 193,678-680. doi:10.1126/science.948742

Charron, A. J. and Sibley, L. D. (2004). Molecular partitioning during host cellpenetration by Toxoplasma gondii. Traffic 5, 855-867. doi:10.1111/j.1600-0854.2004.00228.x

Chauhan, P., Shukla, D., Chattopadhyay, D. and Saha, B. (2017). Redundant andregulatory roles for Toll-like receptors in Leishmania infection.Clin. Exp. Immunol.190, 167-186. doi:10.1111/cei.13014

Chen, L., Lopaticki, S., Riglar, D. T., Dekiwadia, C., Uboldi, A. D., Tham, W. H.,O’Neill, M. T., Richard, D., Baum, J., Ralph, S. A. et al. (2011). An egf-likeprotein forms a complex with pfrh5 and is required for invasion of humanerythrocytes by plasmodium falciparum.PLoS Pathog. 7, e1002199. doi:10.1371/annotation/59703f7f-9506-49d1-b339-09ee31510e89

Coleman, B. I., Saha, S., Sato, S., Engelberg, K., Ferguson, D. J. P., Coppens, I.,Lodoen, M. B. and Gubbels, M.-J. (2018). A member of the ferlin calcium sensorfamily is essential for toxoplasma gondii rhoptry secretion. MBio 9, e01510-18.doi:10.1128/mBio.01510-18

Combs, T. P., Nagajyothi, Mukherjee, S., De Almeida, C. J. G., Jelicks, L. A.,Schubert, W., Lin, Y., Jayabalan, D. S., Zhao, D., Braunstein, V. L. et al. (2005).The adipocyte as an important target cell for Trypanosoma cruzi infection. J. Biol.Chem. 280, 24085-24094. doi:10.1074/jbc.M412802200

Coppi, A., Pinzon-Ortiz, C., Hutter, C. and Sinnis, P. (2005). The Plasmodiumcircumsporozoite protein is proteolytically processed during cell invasion. J. Exp.Med. 201, 27-33. doi:10.1084/jem.20040989

Corrotte, M. and Castro-Gomes, T. (2019). Lysosomes and plasma membranerepair. In Current Topics in Membranes (Ed. Andrade, L.O.), pp. 1-16. AcademicPress Inc.

Cortez, C., Real, F. and Yoshida, N. (2016). Lysosome biogenesis/scatteringincreases host cell susceptibility to invasion by Trypanosoma cruzi metacyclicforms and resistance to tissue culture trypomastigotes. Cell. Microbiol. 18,748-760. doi:10.1111/cmi.12548

Cossart, P. and Helenius, A. (2014). Endocytosis of viruses and bacteria. ColdSpring Harb. Perspect. Biol. 6, a016972. doi:10.1101/cshperspect.a016972

Counihan, N. A., Kalanon, M., Coppel, R. L. and de Koning-Ward, T. F. (2013).Plasmodium rhoptry proteins: why order is important. Trends Parasitol. 29,228-236. doi:10.1016/j.pt.2013.03.003

Coura, J. R. and Dias, J. C. P. (2009). Epidemiology, control and surveillance ofChagas disease: 100 years after its discovery. Mem. Inst. Oswaldo Cruz 104Suppl. 1, 31-40. doi:10.1590/S0074-02762009000900006

Courret, N., Fréhel, C., Gouhier, N., Pouchelet, M., Prina, E., Roux, P. andAntoine, J.-C. (2002). Biogenesis of Leishmania-harbouring parasitophorousvacuoles following phagocytosis of the metacyclic promastigote or amastigotestages of the parasites. J. Cell Sci. 115, 2303-2316.

Cowman, A. F. and Crabb, B. S. (2006). Invasion of red blood cells by malariaparasites. Cell 124, 755-766. doi:10.1016/j.cell.2006.02.006

Cowman, A. F., Tonkin, C. J., Tham, W. H. and Duraisingh, M. T. (2017). Themolecular basis of erythrocyte invasion by malaria parasites. Cell Host Microbe22, 232-245. doi:10.1016/j.chom.2017.07.003

Crosnier, C., Bustamante, L. Y., Bartholdson, S. J., Bei, A. K., Theron, M.,Uchikawa, M., Mboup, S., Ndir, O., Kwiatkowski, D. P., Duraisingh, M. T. et al.(2011). Basigin is a receptor essential for erythrocyte invasion by Plasmodiumfalciparum. Nature 480, 534-537. doi:10.1038/nature10606

Curtidor, H., Patin ̃o, L. C., Arévalo-Pinzón, G., Patarroyo, M. E. and Patarroyo,M. A. (2011). Identification of the Plasmodium falciparum rhoptry neck protein 5(PfRON5). Gene 474, 22-28. doi:10.1016/j.gene.2010.12.005

Da Silva, R. P., Hall, B. F., Joiner, K. A. and Sacks, D. L. (1989). CR1, the C3breceptor, mediates binding of infective Leishmania major metacyclicpromastigotes to human macrophages. J. Immunol. 143, 617-622.

Dasgupta, S., Auth, T., Gov, N. S., Satchwell, T. J., Hanssen, E., Zuccala, E. S.,Riglar, D. T., Toye, A. M., Betz, T., Baum, J. et al. (2014). Membrane-wrappingcontributions to malaria parasite invasion of the human erythrocyte. Biophys. J.107, 43-54. doi:10.1016/j.bpj.2014.05.024

Davies, E. V., Singleton, A. M. and Blackwell, J. M. (1988). Differences in Lshgene control over systemic Leishmania major and Leishmania donovani orLeishmania mexicana mexicana infections are caused by differential targeting toinfiltrating and resident liver macrophage populations. Infect. Immun. 56,1128-1134. doi:10.1128/IAI.56.5.1128-1134.1988

Dawn, A., Singh, S., More, K. R., Siddiqui, F. A., Pachikara, N., Ramdani, G.,Langsley, G. and Chitnis, C. E. (2014). The central role of cAMP in regulatingplasmodium falciparum merozoite invasion of human erythrocytes. PLoS Pathog.10, e1004520. doi:10.1371/journal.ppat.1004520

de Carvalho, T. M. U. and de Souza, and W. , (1989). Early events related with thebehaviour of Trypanosoma cruzi within an endocytic vacuole in mouse peritonealmacrophages. Cell Struct. Funct. 14, 383-392. doi:10.1247/csf.14.383

De Freitas Balanco, J. M., Costa Moreira, M. E., Bonomo, A., Bozza, P. T.,Amarante-Mendes,G., Pirmez,C. andBarcinski,M.A. (2001). Apoptoticmimicryby an obligate intracellular parasite downregulates macrophage microbicidalactivity. Curr. Biol. 11, 1870-1873. doi:10.1016/S0960-9822(01)00563-2

11


Journal

ofCe

llScience

https://doi.org/10.1038/sj.emboj.7601704https://doi.org/10.1038/sj.emboj.7601704https://doi.org/10.1046/j.1365-3024.2002.00486.xhttps://doi.org/10.1046/j.1365-3024.2002.00486.xhttps://doi.org/10.1046/j.1365-3024.2002.00486.xhttps://doi.org/10.1046/j.1365-3024.2002.00486.xhttps://doi.org/10.1084/jem.191.12.2121https://doi.org/10.1084/jem.191.12.2121https://doi.org/10.1084/jem.191.12.2121https://doi.org/10.1016/j.jsb.2015.02.008https://doi.org/10.1016/j.jsb.2015.02.008https://doi.org/10.1182/blood-2009-09-243725https://doi.org/10.1182/blood-2009-09-243725https://doi.org/10.1182/blood-2009-09-243725https://doi.org/10.1182/blood-2009-09-243725https://doi.org/10.1074/jbc.M504158200https://doi.org/10.1074/jbc.M504158200https://doi.org/10.1074/jbc.M504158200https://doi.org/10.1074/jbc.M504158200https://doi.org/10.1074/jbc.M504158200https://doi.org/10.1080/00034983.1984.11811781https://doi.org/10.1080/00034983.1984.11811781https://doi.org/10.1080/00034983.1984.11811781https://doi.org/10.1074/jbc.M008294200https://doi.org/10.1074/jbc.M008294200https://doi.org/10.1074/jbc.M008294200https://doi.org/10.1074/jbc.M008294200https://doi.org/10.1146/annurev.mi.27.100173.002023https://doi.org/10.1146/annurev.mi.27.100173.002023https://doi.org/10.1016/0169-4758(95)80054-9https://doi.org/10.1016/0169-4758(95)80054-9https://doi.org/10.1016/0169-4758(95)80054-9https://doi.org/10.1016/0169-4758(95)80054-9https://doi.org/10.1128/IAI.67.9.4477-4484.1999https://doi.org/10.1128/IAI.67.9.4477-4484.1999https://doi.org/10.1128/IAI.67.9.4477-4484.1999https://doi.org/10.1074/jbc.R000005200https://doi.org/10.1074/jbc.R000005200https://doi.org/10.1074/jbc.R000005200https://doi.org/10.1016/j.chom.2016.02.006https://doi.org/10.1016/j.chom.2016.02.006https://doi.org/10.1016/j.chom.2016.02.006https://doi.org/10.1016/j.chom.2016.02.006https://doi.org/10.1074/jbc.270.10.5172https://doi.org/10.1074/jbc.270.10.5172https://doi.org/10.1074/jbc.270.10.5172https://doi.org/10.1074/jbc.270.10.5172https://doi.org/10.1074/jbc.270.10.5172https://doi.org/10.1111/j.1574-6968.2009.01799.xhttps://doi.org/10.1111/j.1574-6968.2009.01799.xhttps://doi.org/10.1111/j.1574-6968.2009.01799.xhttps://doi.org/10.1093/emboj/17.17.4975https://doi.org/10.1093/emboj/17.17.4975https://doi.org/10.1093/emboj/17.17.4975https://doi.org/10.3389/fimmu.2012.00327https://doi.org/10.3389/fimmu.2012.00327https://doi.org/10.3389/fimmu.2012.00327https://doi.org/10.3389/fimmu.2012.00327https://doi.org/10.1016/j.parint.2008.09.005https://doi.org/10.1016/j.parint.2008.09.005https://doi.org/10.1016/j.parint.2008.09.005https://doi.org/10.1016/j.parint.2008.09.005https://doi.org/10.1016/j.mib.2006.06.017https://doi.org/10.1016/j.mib.2006.06.017https://doi.org/10.1016/j.mib.2006.06.017https://doi.org/10.1242/jcs.226183https://doi.org/10.1242/jcs.226183https://doi.org/10.1242/jcs.226183https://doi.org/10.1242/jcs.226183https://doi.org/10.1242/jcs.226183https://doi.org/10.1093/emboj/21.11.2526https://doi.org/10.1093/emboj/21.11.2526https://doi.org/10.1093/emboj/21.11.2526https://doi.org/10.1023/a:1020192512001https://doi.org/10.1023/a:1020192512001https://doi.org/10.1023/a:1020192512001https://doi.org/10.1126/science.948742https://doi.org/10.1126/science.948742https://doi.org/10.1126/science.948742https://doi.org/10.1111/j.1600-0854.2004.00228.xhttps://doi.org/10.1111/j.1600-0854.2004.00228.xhttps://doi.org/10.1111/j.1600-0854.2004.00228.xhttps://doi.org/10.1111/cei.13014https://doi.org/10.1111/cei.13014https://doi.org/10.1111/cei.13014https://doi.org/10.1371/annotation/59703f7f-9506-49d1-b339-09ee31510e89https://doi.org/10.1371/annotation/59703f7f-9506-49d1-b339-09ee31510e89https://doi.org/10.1371/annotation/59703f7f-9506-49d1-b339-09ee31510e89https://doi.org/10.1371/annotation/59703f7f-9506-49d1-b339-09ee31510e89https://doi.org/10.1371/annotation/59703f7f-9506-49d1-b339-09ee31510e89https://doi.org/10.1128/mBio.01510-18https://doi.org/10.1128/mBio.01510-18https://doi.org/10.1128/mBio.01510-18https://doi.org/10.1128/mBio.01510-18https://doi.org/10.1074/jbc.M412802200https://doi.org/10.1074/jbc.M412802200https://doi.org/10.1074/jbc.M412802200https://doi.org/10.1074/jbc.M412802200https://doi.org/10.1084/jem.20040989https://doi.org/10.1084/jem.20040989https://doi.org/10.1084/jem.20040989https://doi.org/10.1111/cmi.12548https://doi.org/10.1111/cmi.12548https://doi.org/10.1111/cmi.12548https://doi.org/10.1111/cmi.12548https://doi.org/10.1101/cshperspect.a016972https://doi.org/10.1101/cshperspect.a016972https://doi.org/10.1016/j.pt.2013.03.003https://doi.org/10.1016/j.pt.2013.03.003https://doi.org/10.1016/j.pt.2013.03.003https://doi.org/10.1590/S0074-02762009000900006https://doi.org/10.1590/S0074-02762009000900006https://doi.org/10.1590/S0074-02762009000900006https://doi.org/10.1016/j.cell.2006.02.006https://doi.org/10.1016/j.cell.2006.02.006https://doi.org/10.1016/j.chom.2017.07.003https://doi.org/10.1016/j.chom.2017.07.003https://doi.org/10.1016/j.chom.2017.07.003https://doi.org/10.1038/nature10606https://doi.org/10.1038/nature10606https://doi.org/10.1038/nature10606https://doi.org/10.1038/nature10606https://doi.org/10.1016/j.gene.2010.12.005https://doi.org/10.1016/j.gene.2010.12.005https://doi.org/10.1016/j.gene.2010.12.005https://doi.org/10.1016/j.bpj.2014.05.024https://doi.org/10.1016/j.bpj.2014.05.024https://doi.org/10.1016/j.bpj.2014.05.024https://doi.org/10.1016/j.bpj.2014.05.024https://doi.org/10.1128/IAI.56.5.1128-1134.1988https://doi.org/10.1128/IAI.56.5.1128-1134.1988https://doi.org/10.1128/IAI.56.5.1128-1134.1988https://doi.org/10.1128/IAI.56.5.1128-1134.1988https://doi.org/10.1128/IAI.56.5.1128-1134.1988https://doi.org/10.1371/journal.ppat.1004520https://doi.org/10.1371/journal.ppat.1004520https://doi.org/10.1371/journal.ppat.1004520https://doi.org/10.1371/journal.ppat.1004520https://doi.org/10.1247/csf.14.383https://doi.org/10.1247/csf.14.383https://doi.org/10.1247/csf.14.383https://doi.org/10.1016/S0960-9822(01)00563-2https://doi.org/10.1016/S0960-9822(01)00563-2https://doi.org/10.1016/S0960-9822(01)00563-2https://doi.

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