The vertebrate central nervous system (CNS) is protected by restrictive cellular barriers that maintain homeostasis and regulate passage of molecules and cells to the brain parenchyma (Abbott et al., 2006). Three main cellular barrier systems protect neurons from blood-borne external insults, such as infection: the blood-brain barrier (BBB), the blood-cerebrospinal fluid barriers and the meningeal barriers (Coureuil et al., 2017). Of these, the BBB exerts the greatest immediate impact on the cerebral microenvironment.

The BBB is anatomically localized to the cerebral microvasculature, which is constituted by capillaries with a luminal diameter (Ø) of <10 μm, arterioles and venules (~10–100 μm Ø) (Wilhelm et al., 2016; Gould et al., 2017). The microvascular lumen is covered by highly specialized endothelial cells characterized by low permeability, low pinocytic activity and high transcellular electrical resistance (TCER) (Stamatovic et al., 2008). Basal lamina, astrocyte ‘end-feet’ and pericytes also contribute to restrict permeability (Daneman and Prat, 2015; Armulik et al., 2010). Intercellular tight junction (TJ) proteins seal the spaces between adjacent endothelial cells and separate the apical from the basolateral side. This restricts the paracellular flux of hydrophilic molecules and regulates the migration of cells across the endothelial barrier (Alon and van Buul, 2017). Focal adhesion kinase (FAK), also named protein tyrosine kinase 2 (PTK2), regulates signaling pathways involved in cell-cell adhesion and focal adhesion complexes in endothelial cells (Lee et al., 2010; Siu et al., 2009). The turnover of FAK has been shown to regulate barrier function, and dysregulation of FAK impacts the integrity of cellular barriers (Ivey et al., 2009) by increasing paracellular permeability, which can imply disruption of the barrier function (Ma et al., 2013).

Inflammation can cause impairment of the blood-CNS barrier functions (Abbott et al., 2006). Immune activity by resident cells and infiltrating leukocytes are crucial to eradicate pathogens that succeed in translocating across the restrictive CNS barriers (Coureuil et al., 2017), but this response can also alter neuronal function (Dando et al., 2014). Therefore, striking a balance between controlling infection and limiting excessive inflammation is crucial (Klein and Hunter, 2017).

The obligate intracellular parasite Toxoplasma gondii infects a broad range of warm-blooded vertebrates, with an estimated 1/3 of the global human population being chronically infected (Pappas et al., 2009). From the point of entry in the intestinal tract, T. gondii rapidly achieves systemic dissemination and establishes latent infection in the CNS (Schlüter and Barragan, 2019). While chronic infection is generally considered asymptomatic, reactivated or acute infection can lead to life-threatening encephalitis in immune-compromised individuals and to neurological disorders in neonates (Montoya and Liesenfeld, 2004). T. gondii tachyzoites use gliding motility to actively invade cells where they replicate (Dobrowolski and Sibley, 1996). Gliding motility also facilitates transmigration of tachyzoites across the endothelium in vitro (Ross et al., 2019; Barragan et al., 2005) and provides an effective mechanism of propulsion in the microenvironment inside tissues (Barragan and Sibley, 2002). In their journey to form chronic cysts, the tachyzoites cross the cellular brain barriers to establish latent infection in the CNS (Feustel et al., 2012).

In recent years, work in the field has focused on parasite dissemination pathways and mechanisms of passage to the brain (Konradt et al., 2016; Drewry et al., 2019; Kanatani et al., 2017; Bhandage et al., 2020; Schneider et al., 2019; Sangaré et al., 2019). Yet, the roles played by the blood-CNS barriers have remained unresolved. Here, we address the implication of the BBB in the early passage of T. gondii tachyzoites to the CNS. Specifically, we assess the loci of passage of T. gondii across the cerebral cellular barriers and the impacts of BBB integrity, dysregulation and inflammation on the access of tachyzoites to the parenchyma. We identify a role for the TJ regulator FAK in the passage of T. gondii to the brain parenchyma.

Passage across the cellular blood-CNS barriers is a prerequisite for the establishment of latent cerebral toxoplasmosis in humans and rodents. Here, we studied the crucial interaction of T. gondii with the brain vasculature in mice.

Our study establishes that the principal point of entry of T. gondii from the blood circulation into the brain parenchyma is at the level of cortical capillaries, similarly in three separate mouse strains and for prototypic T. gondii type I and type II strains. The data evidence preferential localization of parasites to capillaries with low α5 laminin/P-glycoprotein expression ratio, a relative less frequent localization to post-capillary venules, and scarce or absence of localization to penetrating arterioles, pre-capillary arterioles and penetrating venules.

We report that the BBB integrity is paramount to restrict T. gondii infection of the brain parenchyma. First, from day one post-inoculation, the accumulation of T. gondii tachyzoites in the brain was dramatically inferior to that observed in lung and liver, indicating a relative lower permissiveness of the brain vasculature to adhesion or invasive events by T. gondii. Second, the initial vascular localization with replicating parasites within brain endothelium (Konradt et al., 2016), was rapidly accompanied by appearance of vacuoles with replicating parasites in the perivascular spaces and in the cerebral parenchyma. Third, perturbations of BBB permeability by pro-inflammatory, anti-inflammatory stimuli or high doses of T. gondii resulted in elevated parasite loads in the CNS. Finally, pharmacological inhibition or induced gene ablation of the endothelial BBB intercellular junction regulator FAK facilitated parasite passage to the brain parenchyma.

In natural oral infections, relatively low numbers of T. gondii cysts ultimately form in the CNS indicating that passage across the BBB is a relatively rare event, compared with the initial parasite loads in the parenchyma of other peripheral organs, for example, lungs, liver, spleen, or in musculature. We validated an experimental approach that allowed controlled assessment of T. gondii localization in the CNS after intraperitoneal or intravenous inoculation and that yielded parasite load distributions comparable with oral and other intraperitoneal infection models (Konradt et al., 2016; Kanatani et al., 2017; Zenner et al., 1998; Mordue et al., 2001). While the approaches utilized in this and other studies permit reproducible quantifications, they may also entail limitations. An impact on the relative localization of parasites or kinetics of passage cannot be excluded. However, similar BBB localization and invasion patterns were observed for both inoculation routes, irrespective of mouse strain, parasite strain or dose. Importantly, for the vast majority of parasite foci, the clear vicinity to one capillary indicated the putative translocation site to the brain parenchyma. Further, the observation of perivascular parasite vacuoles beneath the vascular endothelium is interesting and the identification of the hosting cells remains to be investigated.

Our data show that T. gondii tachyzoites very early (1–3 dpi) interact primarily with the endothelium of cortical capillaries, confirming and extending the earlier reported replication of parasites in brain endothelium at later timepoints (7–9 dpi) (Konradt et al., 2016). This localization contrasts with findings in cerebral malaria, where parasitized erythrocytes preferentially bind to post-capillary venules and larger venules with components of inflammatory leukocytes (Haldar et al., 2007; Nacer et al., 2014). The capillary bed offers the largest hemodynamic resistance to the cortical blood supply and has lower flow speeds (Gould et al., 2017). Hypothetically, this together with the restricted diameter of capillaries may facilitate the adhesion, invasion and crossing of T. gondii tachyzoites. In vitro, ICAM-1 and integrins are implicated in adhesion and transmigration of T. gondii tachyzoites (Barragan et al., 2005) and of parasitized leukocytes (Ueno et al., 2014; Ross et al., 2021). Among other inflammatory parameters and cell adhesion molecules (CAMs), here we report elevated expression of ICAM-1 and VCAM-1 in infected brains and, specifically, in purified brain microvessels from challenged mice. Jointly with the finding of elevated TIMP-1 expression in microvessels, and its recently attributed functions in maintenance of FAK-related BBB integrity (Tang et al., 2020) and T. gondii dissemination (Ólafsson et al., 2018; Ólafsson et al., 2019), future investigations need to address the implication of CAMs in endothelial and parenchymal invasion.

We provide evidence that T. gondii can invade the CNS across the choroid plexus (CP) and meningeal vessels, albeit in lower relative numbers compared with total parasite numbers entering across the extensive parenchymal vascular network. However, taking into account the relative smaller endothelial cell surface area and tissue volume of the CP and meningeal vessels related to cortical vasculature, the data indicate that passage is facilitated at the CP. Hypothetically, this may implicate the fenestrated choroidal endothelium, a known locus minoris resistentiae for brain invasion by the causative agent of sleeping sickness Trypanosoma brucei (Schultzberg et al., 1988). Similarly, bacteria and viruses can translocate across meninges and CP (Schwerk et al., 2015) or by disrupting the parenchymal BBB (Doran et al., 2016; Spindler and Hsu, 2012). The data is also in line with the reported localization of T. gondii reactivation foci in grey matter, with few reactivation foci in the CP and the meninges (Dellacasa-Lindberg et al., 2007). Further, human cerebral toxoplasmosis is associated with encephalitis and seldomly associated with meningeal inflammation (Schlüter and Barragan, 2019), while detection of T. gondii in the cerebrospinal fluid is used in clinical practice (Mesquita et al., 2010). Jointly, this indicates that, while parenchymal invasion occurs primarily across cortical capillaries, passage across the CP and meningeal vessels provides T. gondii access to the cerebrospinal fluid circulatory system, which may contribute to parasite dissemination within the CNS.

We report that early passage of T. gondii to the brain parenchyma occurs in absence of measurable BBB leakage, perivascular cuffing or prominent leukocyte infiltration, similarly in different mouse strains and for T. gondii type I and type II strains. In fact, measurable leukocyte infiltration in the vicinity of parasite foci was absent early during infection (< day 5). In contrast, infiltration was abundant by day 10, in line with recent findings (Schneider et al., 2019). Thus, perivascular cuffing or significant leukocyte infiltration does not appear to be a requisite for initial T. gondii passage to the parenchyma (< day 5). However, an inflammatory activation of the BBB endothelium by the infection was evidenced by elevated expression of CAMs but also inflammation-dampening TIMP-1 (Ólafsson et al., 2018; Ólafsson et al., 2019). Pro-inflammatory pre-treatment with LPS yielded elevations of parenchymal parasite loads and significantly larger cerebral parasite foci. This indicates that initial parasite passage was facilitated by LPS-induced increased BBB permeability (Banks et al., 2015), which may implicate regulation via FAK (Guo et al., 2015), and by exacerbated inflammation (Haruwaka et al., 2019). Conversely, anti-inflammatory pre-treatment with hydrocortisone led to elevated total parasite loads in the brain but also in other organs, likely secondary to a hampered immune response. Jointly, though these treatments have pleiotropic effects with a likely impact on parasitemia, the data is indicative that passage of T. gondii across the BBB is not only restricted by the inflammatory response. Along these lines, unnaturally high doses of T. gondii tachyzoites were required to induce BBB leakage similar to LPS. This strongly advocates against a generalized BBB dysfunction early in natural T. gondii infections, but does not exclude the occurrence of focalized dysregulations (Estato et al., 2018). In fact, focalized permeability elevations were detected in the vicinity of parasite foci, albeit in low numbers and without increased relative numbers over time. This is indicative of BBB permeability variations over time following the penetration of parasites to the parenchyma or that the BBB is transiently dysregulated. Thus, future investigations need to determine if an elevated BBB permeability around a given focus is consistently present, albeit transient, or if translocation can also occur in the absence of elevated permeability. Also, taking into consideration the alternative pathways of translocation discussed below, these two possibilities are not necessarily mutually exclusive. Noteworthy, despite the infection of endothelium and expected natural lysis of parasitized cells after replication, we found no evidence of irreversible vascular disruption with petechial hemorrhages, as described in cerebral malaria (Haldar et al., 2007) and meningococcal disease (Coureuil et al., 2014). Yet, this does not rule out that single translocation events of T. gondii or egress of tachyzoites from single endothelial cells transiently destabilize the BBB below the detection threshold for the permeability tracer EB. In in vitro models using polarized primary brain endothelium, extracellular tachyzoites and parasitized dendritic cells rapidly translocated in absence of endothelial cell lysis, with a modest perturbation of the transcellular electrical resistance (TCER) but, importantly, in absence of leakage of a low-molecular weight permeability tracer (Ross et al., 2019; Ross et al., 2021). In sharp contrast, cellular depolarization events or lysis of cells in the monolayers following parasite replication at later time-points led to significant decreases in TCER and dramatic elevations of permeability tracer concentration in vitro (Ross et al., 2019; Ross et al., 2021). Altogether, these findings motivated an exploration of the BBB regulatory mechanisms in vivo.

We demonstrate a role for the BBB junction regulator FAK in the restricted passage of T. gondii to the brain parenchyma. Despite being embryonic lethal (Ilić et al., 1995), unchallenged adult mice with conditional deletion of endothelial FAK normally exhibit wildtype phenotype and apparently normal BBB morphology (Lee et al., 2010; Weis et al., 2008), consistent with the maintained morphology and the absence of leakage of blood tracer observed here. However, upon T. gondii challenge, FAK-deficient mice carried higher numbers of parasite foci and foci of larger size in the parenchyma, indicative of increased permissiveness compared to wildtype. Interestingly, pharmacological treatment with FAK inhibitor had a more pronounced effect on parasite loads with elevated BBB permeability, compared with conditional ablation of endothelial FAK. This difference may be due to a number of factors. First, conditional deletion of endothelial FAK is partially functionally compensated (Weis et al., 2008). Second, although the FAK inhibitor defactinib appears to be well tolerated, immunomodulatory effects may also contribute to the higher parasite loads in peripheral organs, and, thus, elevated numbers of parasites may enter the cerebral circulation (Osipov et al., 2019). Additionally, inhibition of FAK may interfere with parasite dissemination in infected leukocytes (Ólafsson et al., 2019; Cook et al., 2018) and parasite survival in endothelium (Portillo et al., 2017). Regardless, induced ablation of FAK in endothelium, with maintained expression of FAK in bone-marrow-derived cells, yielded higher numbers and size of parasite foci in brain parenchyma, demonstrating endothelium-related effects. Further, we recently reported that altered FAK phosphorylation upon T. gondii infection facilitates parasite passage across highly polarized primary brain endothelial cell monolayers (Ross et al., 2019). Importantly, this transient dysregulation of FAK occurs without disruption of monolayer polarization or increased permeability to low-molecular weight tracer, consistent with in vivo observations here. Thus, FAK inhibition or ablation appears to have a destabilizing, but non-disruptive, effect on the BBB. Jointly, the data show that endothelial FAK-regulated BBB restrictiveness is one of the determinants of T. gondii passage across the BBB.

Alternative routes have been proposed for the translocation of T. gondii across restrictive biological barriers (Lambert and Barragan, 2010), including the BBB (Schlüter and Barragan, 2019), and it is likely that more than one mechanism mediates entry into the CNS, including growth across endothelium, direct penetration and trafficking of leukocytes (Matta et al., 2021). The paracellular entry route (Ross et al., 2019; Barragan et al., 2005; Barragan and Sibley, 2002; Furtado et al., 2012; Weight et al., 2015) implies passing cellular TJs, and the transcellular route (Konradt et al., 2016; Lambert and Barragan, 2010; Dubey, 1997) implies apical parasite invasion and basolateral exit from the cells after replication. Additionally, leukocytes traffic into the brain parenchyma during toxoplasmosis (John et al., 2011) and parasitized hypermigratory leukocytes may aid in direct translocation (Bhandage et al., 2020; Courret et al., 2006; Lachenmaier et al., 2011) or by depositing parasites on the endothelium (Lambert and Barragan, 2010; Baba et al., 2017). Although the relative importance of the alternative pathways of passage is not specifically addressed here, the data are relevant for these alternatives or combinations of pathways because focal invasion events with an impact on TJ and BBB barrier integrity are likely to occur, for example, upon egress of tachyzoites from endothelium (Konradt et al., 2016). In these settings, we demonstrate that a non-replicating T. gondii line (CPS) transmigrates across polarized endothelium and epithelium in vitro, with elevated transmigration frequencies upon FAK inhibition. Thus, despite the reduced gliding motility and transmigration abilities of this mutant, the data provide additional evidence that FAK impacts the endothelial process of tachyzoite transmigration, in absence of monolayer disruption and independently of parasite replication in vitro. In mice, surprisingly few CPS parasites were detected in the brain and other peripheral organs shortly after inoculation, consistent with the absence of symptomatology in mice despite the inoculation of doses that are imminently lethal with wildtype ( > 10⁸) and with scarce localization of single CPS tachyzoites intravascularly, and extravascularly in the brain parenchyma. Partly contrasting with in vitro results, elevated total numbers of CPS parasites were not detected in the brain upon FAK inhibition or ablation in vivo. Given the high TCER of the BBB, this may be related to the expected overall reduced invasive and transmigration abilities of this mutant, in combination with a sensitivity limitation of the assay due to scarce total invasion events. Importantly, the low numbers of intravascular CPS parasites compared with wildtype (RH) precluded direct comparisons with wildtype for numbers of translocation events in vivo. Together, the reduced gliding and TCER-related transmigration abilities of this auxotroph mutant upon uracil deprivation indicate that metabolic stress (Blondy et al., 2020) negatively impacts invasion events in vitro and in vivo and, that this mutant is rapidly neutralized in vivo, possibly as a consequence of its generally reduced invasion capability.

Jointly, the present study identifies FAK-regulated TJ stability as a determinant of T. gondii translocation across cortical capillaries. We postulate that the infection-related destabilization of the BBB by FAK dephosphorylation (Ross et al., 2019) facilitates active transmigration of parasites across the BBB. The impact of transient and focal BBB dysregulation on the alternative pathways of parasite passage to the parenchyma, together with the roles of the non-endothelial cellular constituents of the neurovascular unit, need to be addressed in future investigations.

In humans and mice, primary infection with T. gondii entails invasion of the CNS with passage of the parasite across the BBB. Natural infections in humans normally present mild or no symptomatology (Montoya and Liesenfeld, 2004). Thus, our data supports the concept that passage of T. gondii to the brain parenchyma is an early and clinically silent process in absence of focal neurological symptoms or cardinal signs of inflammation. As the immune response develops, tachyzoites are neutralized and developmental switch to bradyzoite stages takes place for chronic perseverance in the brain parenchyma.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Abbott NJ
   2. Rönnbäck L
   3. Hansson E

   (2006) Astrocyte-endothelial interactions at the blood-brain barrier

   Nature Reviews. Neuroscience 7:41–53.

   https://doi.org/10.1038/nrn1824

   • PubMed
   • Google Scholar
2. 1. Alon R
   2. van Buul JD

   (2017) Leukocyte Breaching of Endothelial Barriers: The Actin Link

   Trends in Immunology 38:606–615.

   https://doi.org/10.1016/j.it.2017.05.002

   • PubMed
   • Google Scholar
3. 1. Armulik A
   2. Genové G
   3. Mäe M
   4. Nisancioglu MH
   5. Wallgard E
   6. Niaudet C
   7. He L
   8. Norlin J
   9. Lindblom P
   10. Strittmatter K
   11. Johansson BR
   12. Betsholtz C

   (2010) Pericytes regulate the blood-brain barrier

   Nature 468:557–561.

   https://doi.org/10.1038/nature09522

   • PubMed
   • Google Scholar
4. 1. Baba M
   2. Batanova T
   3. Kitoh K
   4. Takashima Y

   (2017) Adhesion of Toxoplasma gondii tachyzoite-infected vehicle leukocytes to capillary endothelial cells triggers timely parasite egression

   Scientific Reports 7:5675.

   https://doi.org/10.1038/s41598-017-05956-z

   • PubMed
   • Google Scholar
5. 1. Banks WA
   2. Gray AM
   3. Erickson MA
   4. Salameh TS
   5. Damodarasamy M
   6. Sheibani N
   7. Meabon JS
   8. Wing EE
   9. Morofuji Y
   10. Cook DG
   11. Reed MJ

   (2015) Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit

   Journal of Neuroinflammation 12:223.

   https://doi.org/10.1186/s12974-015-0434-1

   • PubMed
   • Google Scholar
6. 1. Barragan A
   2. Sibley LD

   (2002) Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence

   The Journal of Experimental Medicine 195:1625–1633.

   https://doi.org/10.1084/jem.20020258

   • PubMed
   • Google Scholar
7. 1. Barragan A
   2. Brossier F
   3. Sibley LD

   (2005) Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2

   Cellular Microbiology 7:561–568.

   https://doi.org/10.1111/j.1462-5822.2005.00486.x

   • PubMed
   • Google Scholar
8. 1. Bhandage AK
   2. Olivera GC
   3. Kanatani S
   4. Thompson E
   5. Loré K
   6. Varas-Godoy M
   7. Barragan A

   (2020) A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites

   eLife 9:e60528.

   https://doi.org/10.7554/eLife.60528

   • PubMed
   • Google Scholar
9. 1. Blondy S
   2. David V
   3. Verdier M
   4. Mathonnet M
   5. Perraud A
   6. Christou N

   (2020) 5-Fluorouracil resistance mechanisms in colorectal cancer: From classical pathways to promising processes

   Cancer Science 111:3142–3154.

   https://doi.org/10.1111/cas.14532

   • PubMed
   • Google Scholar
10. 1. Cook JH
    2. Ueno N
    3. Lodoen MB

    (2018) Toxoplasma gondii disrupts β1 integrin signaling and focal adhesion formation during monocyte hypermotility

    The Journal of Biological Chemistry 293:3374–3385.

    https://doi.org/10.1074/jbc.M117.793281

    • PubMed
    • Google Scholar
11. 1. Coureuil M
    2. Bourdoulous S
    3. Marullo S
    4. Nassif X

    (2014) Invasive meningococcal disease: a disease of the endothelial cells

    Trends in Molecular Medicine 20:571–578.

    https://doi.org/10.1016/j.molmed.2014.08.002

    • PubMed
    • Google Scholar
12. 1. Coureuil M
    2. Lécuyer H
    3. Bourdoulous S
    4. Nassif X

    (2017) A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers

    Nature Reviews. Microbiology 15:149–159.

    https://doi.org/10.1038/nrmicro.2016.178

    • PubMed
    • Google Scholar
13. 1. Courret N
    2. Darche S
    3. Sonigo P
    4. Milon G
    5. Buzoni-Gâtel D
    6. Tardieux I

    (2006) CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain

    Blood 107:309–316.

    https://doi.org/10.1182/blood-2005-02-0666

    • PubMed
    • Google Scholar
14. 1. Dando SJ
    2. Mackay-Sim A
    3. Norton R
    4. Currie BJ
    5. St John JA
    6. Ekberg JAK
    7. Batzloff M
    8. Ulett GC
    9. Beacham IR

    (2014) Pathogens penetrating the central nervous system: infection pathways and the cellular and molecular mechanisms of invasion

    Clinical Microbiology Reviews 27:691–726.

    https://doi.org/10.1128/CMR.00118-13

    • PubMed
    • Google Scholar
15. 1. Daneman R
    2. Prat A

    (2015) The blood-brain barrier

    Cold Spring Harbor Perspectives in Biology 7:a020412.

    https://doi.org/10.1101/cshperspect.a020412

    • PubMed
    • Google Scholar
16. 1. Dellacasa-Lindberg I
    2. Hitziger N
    3. Barragan A

    (2007) Localized recrudescence of Toxoplasma infections in the central nervous system of immunocompromised mice assessed by in vivo bioluminescence imaging

    Microbes and Infection 9:1291–1298.

    https://doi.org/10.1016/j.micinf.2007.06.003

    • PubMed
    • Google Scholar
17. 1. Dellacasa-Lindberg I
    2. Fuks JM
    3. Arrighi RBG
    4. Lambert H
    5. Wallin RPA
    6. Chambers BJ
    7. Barragan A

    (2011) Migratory activation of primary cortical microglia upon infection with Toxoplasma gondii

    Infection and Immunity 79:3046–3052.

    https://doi.org/10.1128/IAI.01042-10

    • PubMed
    • Google Scholar
18. 1. Derouin F
    2. Garin YJF

    (1991) Toxoplasma gondii: blood and tissue kinetics during acute and chronic infections in mice

    Experimental Parasitology 73:460–468.

    https://doi.org/10.1016/0014-4894(91)90070-d

    • PubMed
    • Google Scholar
19. 1. Dobrowolski JM
    2. Sibley LD

    (1996) Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite

    Cell 84:933–939.

    https://doi.org/10.1016/s0092-8674(00)81071-5

    • PubMed
    • Google Scholar
20. 1. Doran KS
    2. Fulde M
    3. Gratz N
    4. Kim BJ
    5. Nau R
    6. Prasadarao N
    7. Schubert-Unkmeir A
    8. Tuomanen EI
    9. Valentin-Weigand P

    (2016) Host-pathogen interactions in bacterial meningitis

    Acta Neuropathologica 131:185–209.

    https://doi.org/10.1007/s00401-015-1531-z

    • PubMed
    • Google Scholar
21. 1. Drewry LL
    2. Jones NG
    3. Wang Q
    4. Onken MD
    5. Miller MJ
    6. Sibley LD

    (2019) The secreted kinase ROP17 promotes Toxoplasma gondii dissemination by hijacking monocyte tissue migration

    Nature Microbiology 4:1951–1963.

    https://doi.org/10.1038/s41564-019-0504-8

    • PubMed
    • Google Scholar
22. 1. Dubey JP

    (1997) Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii

    The Journal of Eukaryotic Microbiology 44:592–602.

    https://doi.org/10.1111/j.1550-7408.1997.tb05965.x

    • PubMed
    • Google Scholar
23. 1. Estato V
    2. Stipursky J
    3. Gomes F
    4. Mergener TC
    5. Frazão-Teixeira E
    6. Allodi S
    7. Tibiriçá E
    8. Barbosa HS
    9. Adesse D

    (2018) The Neurotropic Parasite Toxoplasma gondii Induces Sustained Neuroinflammation with Microvascular Dysfunction in Infected Mice

    The American Journal of Pathology 188:2674–2687.

    https://doi.org/10.1016/j.ajpath.2018.07.007

    • PubMed
    • Google Scholar
24. 1. Feustel SM
    2. Meissner M
    3. Liesenfeld O

    (2012) Toxoplasma gondii and the blood-brain barrier

    Virulence 3:182–192.

    https://doi.org/10.4161/viru.19004

    • PubMed
    • Google Scholar
25. 1. Fox BA
    2. Bzik DJ

    (2002) De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii

    Nature 415:926–929.

    https://doi.org/10.1038/415926a

    • PubMed
    • Google Scholar
26. 1. Fuks JM
    2. Arrighi RBG
    3. Weidner JM
    4. Kumar Mendu S
    5. Jin Z
    6. Wallin RPA
    7. Rethi B
    8. Birnir B
    9. Barragan A

    (2012) GABAergic signaling is linked to a hypermigratory phenotype in dendritic cells infected by Toxoplasma gondii

    PLOS Pathogens 8:e1003051.

    https://doi.org/10.1371/journal.ppat.1003051

    • PubMed
    • Google Scholar
27. 1. Furtado JM
    2. Bharadwaj AS
    3. Chipps TJ
    4. Pan Y
    5. Ashander LM

    (2012) Toxoplasma gondii tachyzoites cross retinal endothelium assisted by intercellular adhesion molecule-1 in vitro

    Immunology and Cell Biology 90:912–915.

    https://doi.org/10.1038/icb.2012.21

    • PubMed
    • Google Scholar
28. 1. Gerber DE
    2. Camidge DR
    3. Morgensztern D
    4. Cetnar J
    5. Kelly RJ
    6. Ramalingam SS
    7. Spigel DR
    8. Jeong W
    9. Scaglioni PP
    10. Zhang S
    11. Li M
    12. Weaver DT
    13. Vaikus L
    14. Keegan M
    15. Horobin JC
    16. Burns TF

    (2020) Phase 2 study of the focal adhesion kinase inhibitor defactinib (VS-6063) in previously treated advanced KRAS mutant non-small cell lung cancer

    Lung Cancer 139:60–67.

    https://doi.org/10.1016/j.lungcan.2019.10.033

    • PubMed
    • Google Scholar
29. 1. Gould IG
    2. Tsai P
    3. Kleinfeld D
    4. Linninger A

    (2017) The capillary bed offers the largest hemodynamic resistance to the cortical blood supply

    Journal of Cerebral Blood Flow and Metabolism 37:52–68.

    https://doi.org/10.1177/0271678X16671146

    • PubMed
    • Google Scholar
30. 1. Guo S
    2. Nighot M
    3. Al-Sadi R
    4. Alhmoud T
    5. Nighot P
    6. Ma TY

    (2015) Lipopolysaccharide Regulation of Intestinal Tight Junction Permeability Is Mediated by TLR4 Signal Transduction Pathway Activation of FAK and MyD88

    Journal of Immunology 195:4999–5010.

    https://doi.org/10.4049/jimmunol.1402598

    • PubMed
    • Google Scholar
31. 1. Haldar K
    2. Murphy SC
    3. Milner DA
    4. Taylor TE

    (2007) Malaria: mechanisms of erythrocytic infection and pathological correlates of severe disease

    Annual Review of Pathology 2:217–249.

    https://doi.org/10.1146/annurev.pathol.2.010506.091913

    • PubMed
    • Google Scholar
32. 1. Haruwaka K
    2. Ikegami A
    3. Tachibana Y
    4. Ohno N
    5. Konishi H
    6. Hashimoto A
    7. Matsumoto M
    8. Kato D
    9. Ono R
    10. Kiyama H
    11. Moorhouse AJ
    12. Nabekura J
    13. Wake H

    (2019) Dual microglia effects on blood brain barrier permeability induced by systemic inflammation

    Nature Communications 10:5816.

    https://doi.org/10.1038/s41467-019-13812-z

    • PubMed
    • Google Scholar
33. 1. Howland SW
    2. Gun SY
    3. Claser C
    4. Poh CM
    5. Rénia L

    (2015) Measuring antigen presentation in mouse brain endothelial cells ex vivo and in vitro

    Nature Protocols 10:2016–2026.

    https://doi.org/10.1038/nprot.2015.129

    • PubMed
    • Google Scholar
34. 1. Ilić D
    2. Furuta Y
    3. Kanazawa S
    4. Takeda N
    5. Sobue K
    6. Nakatsuji N
    7. Nomura S
    8. Fujimoto J
    9. Okada M
    10. Yamamoto T

    (1995) Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice

    Nature 377:539–544.

    https://doi.org/10.1038/377539a0

    • PubMed
    • Google Scholar
35. 1. Ivey NS
    2. Renner NA
    3. Moroney-Rasmussen T
    4. Mohan M
    5. Redmann RK
    6. Didier PJ
    7. Alvarez X
    8. Lackner AA
    9. MacLean AG

    (2009) Association of FAK activation with lentivirus-induced disruption of blood-brain barrier tight junction-associated ZO-1 protein organization

    Journal of Neurovirology 15:312–323.

    https://doi.org/10.1080/13550280902998413

    • PubMed
    • Google Scholar
36. 1. John B
    2. Ricart B
    3. Tait Wojno ED
    4. Harris TH
    5. Randall LM
    6. Christian DA
    7. Gregg B
    8. De Almeida DM
    9. Weninger W
    10. Hammer DA
    11. Hunter CA

    (2011) Analysis of behavior and trafficking of dendritic cells within the brain during toxoplasmic encephalitis

    PLOS Pathogens 7:e1002246.

    https://doi.org/10.1371/journal.ppat.1002246

    • PubMed
    • Google Scholar
37. 1. Kanatani S
    2. Fuks JM
    3. Olafsson EB
    4. Westermark L
    5. Chambers B
    6. Varas-Godoy M
    7. Uhlén P
    8. Barragan A

    (2017) Voltage-dependent calcium channel signaling mediates GABAA receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii

    PLOS Pathogens 13:e1006739.

    https://doi.org/10.1371/journal.ppat.1006739

    • PubMed
    • Google Scholar
38. 1. Kanteti R
    2. Mirzapoiazova T
    3. Riehm JJ
    4. Dhanasingh I
    5. Mambetsariev B
    6. Wang J
    7. Kulkarni P
    8. Kaushik G
    9. Seshacharyulu P
    10. Ponnusamy MP
    11. Kindler HL
    12. Nasser MW
    13. Batra SK
    14. Salgia R

    (2018) Focal adhesion kinase a potential therapeutic target for pancreatic cancer and malignant pleural mesothelioma

    Cancer Biology & Therapy 19:316–327.

    https://doi.org/10.1080/15384047.2017.1416937

    • PubMed
    • Google Scholar
39. 1. Kim K
    2. Eaton MS
    3. Schubert W
    4. Wu S
    5. Tang J

    (2001) Optimized expression of green fluorescent protein in Toxoplasma gondii using thermostable green fluorescent protein mutants

    Molecular and Biochemical Parasitology 113:309–313.

    https://doi.org/10.1016/s0166-6851(01)00212-2

    • PubMed
    • Google Scholar
40. 1. Klein RS
    2. Hunter CA

    (2017) Protective and Pathological Immunity during Central Nervous System Infections

    Immunity 46:891–909.

    https://doi.org/10.1016/j.immuni.2017.06.012

    • PubMed
    • Google Scholar
41. 1. Konradt C
    2. Ueno N
    3. Christian DA
    4. Delong JH
    5. Pritchard GH
    6. Herz J
    7. Bzik DJ
    8. Koshy AA
    9. McGavern DB
    10. Lodoen MB
    11. Hunter CA

    (2016) Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system

    Nature Microbiology 1:16001.

    https://doi.org/10.1038/nmicrobiol.2016.1

    • PubMed
    • Google Scholar
42. 1. Lachenmaier SM
    2. Deli MA
    3. Meissner M
    4. Liesenfeld O

    (2011) Intracellular transport of Toxoplasma gondii through the blood-brain barrier

    Journal of Neuroimmunology 232:119–130.

    https://doi.org/10.1016/j.jneuroim.2010.10.029

    • PubMed
    • Google Scholar
43. 1. Lambert H
    2. Barragan A

    (2010) Modelling parasite dissemination: host cell subversion and immune evasion by Toxoplasma gondii

    Cellular Microbiology 12:292–300.

    https://doi.org/10.1111/j.1462-5822.2009.01417.x

    • PubMed
    • Google Scholar
44. 1. Lee J
    2. Borboa AK
    3. Chun HB
    4. Baird A
    5. Eliceiri BP

    (2010) Conditional deletion of the focal adhesion kinase FAK alters remodeling of the blood-brain barrier in glioma

    Cancer Research 70:10131–10140.

    https://doi.org/10.1158/0008-5472.CAN-10-2740

    • PubMed
    • Google Scholar
45. 1. Ma Y
    2. Semba S
    3. Khan RI
    4. Bochimoto H
    5. Watanabe T
    6. Fujiya M
    7. Kohgo Y
    8. Liu Y
    9. Taniguchi T

    (2013) Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction

    Biochimica et Biophysica Acta 1832:151–159.

    https://doi.org/10.1016/j.bbadis.2012.10.006

    • PubMed
    • Google Scholar
46. 1. Manaenko A
    2. Chen H
    3. Kammer J
    4. Zhang JH
    5. Tang J

    (2011) Comparison Evans Blue injection routes: Intravenous versus intraperitoneal, for measurement of blood-brain barrier in a mice hemorrhage model

    Journal of Neuroscience Methods 195:206–210.

    https://doi.org/10.1016/j.jneumeth.2010.12.013

    • PubMed
    • Google Scholar
47. 1. Masocha W
    2. Robertson B
    3. Rottenberg ME
    4. Mhlanga J
    5. Sorokin L
    6. Kristensson K

    (2004) Cerebral vessel laminins and IFN-gamma define Trypanosoma brucei brucei penetration of the blood-brain barrier

    The Journal of Clinical Investigation 114:689–694.

    https://doi.org/10.1172/JCI22104

    • PubMed
    • Google Scholar
48. 1. Matta SK
    2. Rinkenberger N
    3. Dunay IR
    4. Sibley LD

    (2021) Toxoplasma gondii infection and its implications within the central nervous system

    Nature Reviews. Microbiology 19:467–480.

    https://doi.org/10.1038/s41579-021-00518-7

    • PubMed
    • Google Scholar
49. 1. Mesquita RT
    2. Ziegler ÂP
    3. Hiramoto RM
    4. Vidal JE
    5. Pereira-Chioccola VL

    (2010) Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

    Journal of Medical Microbiology 59:641–647.

    https://doi.org/10.1099/jmm.0.016261-0

    • PubMed
    • Google Scholar
50. 1. Montoya JG
    2. Liesenfeld O

    (2004) Toxoplasmosis

    Lancet 363:1965–1976.

    https://doi.org/10.1016/S0140-6736(04)16412-X

    • PubMed
    • Google Scholar
51. 1. Mordue DG
    2. Monroy F
    3. La Regina M
    4. Dinarello CA
    5. Sibley LD

    (2001) Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines

    Journal of Immunology 167:4574–4584.

    https://doi.org/10.4049/jimmunol.167.8.4574

    • PubMed
    • Google Scholar
52. 1. Nacer A
    2. Movila A
    3. Sohet F
    4. Girgis NM
    5. Gundra UM
    6. Loke P
    7. Daneman R
    8. Frevert U

    (2014) Experimental cerebral malaria pathogenesis--hemodynamics at the blood brain barrier

    PLOS Pathogens 10:e1004528.

    https://doi.org/10.1371/journal.ppat.1004528

    • PubMed
    • Google Scholar
53. 1. Ólafsson EB
    2. Varas-Godoy M
    3. Barragan A

    (2018) Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix

    Cellular Microbiology 20:12808.

    https://doi.org/10.1111/cmi.12808

    • PubMed
    • Google Scholar
54. 1. Ólafsson EB
    2. Ross EC
    3. Varas-Godoy M
    4. Barragan A

    (2019) Correction: TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63-ITGB1-FAK signaling (doi:10.1242/jcs.225193)

    Journal of Cell Science 132:jcs230920.

    https://doi.org/10.1242/jcs.230920

    • PubMed
    • Google Scholar
55. 1. Osipov A
    2. Saung MT
    3. Zheng L
    4. Murphy AG

    (2019) Small molecule immunomodulation: the tumor microenvironment and overcoming immune escape

    Journal for Immunotherapy of Cancer 7:224.

    https://doi.org/10.1186/s40425-019-0667-0

    • PubMed
    • Google Scholar
56. 1. Pappas G
    2. Roussos N
    3. Falagas ME

    (2009) Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis

    International Journal for Parasitology 39:1385–1394.

    https://doi.org/10.1016/j.ijpara.2009.04.003

    • PubMed
    • Google Scholar
57. 1. Portillo J-AC
    2. Muniz-Feliciano L
    3. Lopez Corcino Y
    4. Lee SJ
    5. Van Grol J
    6. Parsons SJ
    7. Schiemman WP
    8. Subauste CS

    (2017) Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy

    PLOS Pathogens 13:e1006671.

    https://doi.org/10.1371/journal.ppat.1006671

    • PubMed
    • Google Scholar
58. 1. Ross EC
    2. Olivera GC
    3. Barragan A

    (2019) Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers

    Cellular Microbiology 21:e13048.

    https://doi.org/10.1111/cmi.13048

    • PubMed
    • Google Scholar
59. 1. Ross EC
    2. Ten Hoeve AL
    3. Barragan A

    (2021) Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers

    Cellular and Molecular Life Sciences 78:5197–5212.

    https://doi.org/10.1007/s00018-021-03858-y

    • PubMed
    • Google Scholar
60. 1. Sangaré LO
    2. Ólafsson EB
    3. Wang Y
    4. Yang N
    5. Julien L
    6. Camejo A
    7. Pesavento P
    8. Sidik SM
    9. Lourido S
    10. Barragan A
    11. Saeij JPJ

    (2019) In Vivo CRISPR Screen Identifies TgWIP as a Toxoplasma Modulator of Dendritic Cell Migration

    Cell Host & Microbe 26:478–492.

    https://doi.org/10.1016/j.chom.2019.09.008

    • Google Scholar
61. 1. Schlüter D
    2. Barragan A

    (2019) Advances and Challenges in Understanding Cerebral Toxoplasmosis

    Frontiers in Immunology 10:242.

    https://doi.org/10.3389/fimmu.2019.00242

    • PubMed
    • Google Scholar
62. 1. Schneider CA
    2. Figueroa Velez DX
    3. Azevedo R
    4. Hoover EM
    5. Tran CJ
    6. Lo C
    7. Vadpey O
    8. Gandhi SP
    9. Lodoen MB

    (2019) Imaging the dynamic recruitment of monocytes to the blood-brain barrier and specific brain regions during Toxoplasma gondii infection

    PNAS 116:24796–24807.

    https://doi.org/10.1073/pnas.1915778116

    • PubMed
    • Google Scholar
63. 1. Schultzberg M
    2. Ambatsis M
    3. Samuelsson EB
    4. Kristensson K
    5. van Meirvenne N

    (1988) Spread of Trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia

    Journal of Neuroscience Research 21:56–61.

    https://doi.org/10.1002/jnr.490210109

    • PubMed
    • Google Scholar
64. 1. Schwerk C
    2. Tenenbaum T
    3. Kim KS
    4. Schroten H

    (2015) The choroid plexus-a multi-role player during infectious diseases of the CNS

    Frontiers in Cellular Neuroscience 9:80.

    https://doi.org/10.3389/fncel.2015.00080

    • PubMed
    • Google Scholar
65. 1. Shen TL
    2. Park AYJ
    3. Alcaraz A
    4. Peng X
    5. Jang I
    6. Koni P
    7. Flavell RA
    8. Gu H
    9. Guan JL

    (2005) Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis

    The Journal of Cell Biology 169:941–952.

    https://doi.org/10.1083/jcb.200411155

    • PubMed
    • Google Scholar
66. 1. Siu ER
    2. Wong EWP
    3. Mruk DD
    4. Porto CS
    5. Cheng CY

    (2009) Focal adhesion kinase is a blood-testis barrier regulator

    PNAS 106:9298–9303.

    https://doi.org/10.1073/pnas.0813113106

    • PubMed
    • Google Scholar
67. 1. Spindler KR
    2. Hsu TH

    (2012) Viral disruption of the blood-brain barrier

    Trends in Microbiology 20:282–290.

    https://doi.org/10.1016/j.tim.2012.03.009

    • PubMed
    • Google Scholar
68. 1. Stamatovic SM
    2. Keep RF
    3. Andjelkovic AV

    (2008) Brain endothelial cell-cell junctions: how to “open” the blood brain barrier

    Current Neuropharmacology 6:179–192.

    https://doi.org/10.2174/157015908785777210

    • PubMed
    • Google Scholar
69. 1. Tang J
    2. Kang Y
    3. Huang L
    4. Wu L
    5. Peng Y

    (2020) TIMP1 preserves the blood-brain barrier through interacting with CD63/integrin β 1 complex and regulating downstream FAK/RhoA signaling

    Acta Pharmaceutica Sinica. B 10:987–1003.

    https://doi.org/10.1016/j.apsb.2020.02.015

    • PubMed
    • Google Scholar
70. 1. Ueno N
    2. Harker KS
    3. Clarke EV
    4. McWhorter FY
    5. Liu WF
    6. Tenner AJ
    7. Lodoen MB

    (2014) Real-time imaging of Toxoplasma-infected human monocytes under fluidic shear stress reveals rapid translocation of intracellular parasites across endothelial barriers

    Cellular Microbiology 16:580–595.

    https://doi.org/10.1111/cmi.12239

    • PubMed
    • Google Scholar
71. 1. Weight CM
    2. Jones EJ
    3. Horn N
    4. Wellner N

    (2015) Elucidating pathways of Toxoplasma gondii invasion in the gastrointestinal tract: involvement of the tight junction protein occludin

    Microbes and Infection 17:698–709.

    https://doi.org/10.1016/j.micinf.2015.07.001

    • PubMed
    • Google Scholar
72. 1. Weis SM
    2. Lim S-T
    3. Lutu-Fuga KM
    4. Barnes LA
    5. Chen XL
    6. Göthert JR
    7. Shen T-L
    8. Guan J-L
    9. Schlaepfer DD
    10. Cheresh DA

    (2008) Compensatory role for Pyk2 during angiogenesis in adult mice lacking endothelial cell FAK

    The Journal of Cell Biology 181:43–50.

    https://doi.org/10.1083/jcb.200710038

    • PubMed
    • Google Scholar
73. 1. Wilhelm I
    2. Nyúl-Tóth Á
    3. Suciu M
    4. Hermenean A
    5. Krizbai IA

    (2016) Heterogeneity of the blood-brain barrier

    Tissue Barriers 4:e1143544.

    https://doi.org/10.1080/21688370.2016.1143544

    • PubMed
    • Google Scholar
74. 1. Xaio H
    2. Banks WA
    3. Niehoff ML
    4. Morley JE

    (2001) Effect of LPS on the permeability of the blood-brain barrier to insulin

    Brain Research 896:36–42.

    https://doi.org/10.1016/s0006-8993(00)03247-9

    • PubMed
    • Google Scholar
75. 1. Zenner L
    2. Darcy F
    3. Capron A
    4. Cesbron-Delauw MF

    (1998) Toxoplasma gondii: kinetics of the dissemination in the host tissues during the acute phase of infection of mice and rats

    Experimental Parasitology 90:86–94.

    https://doi.org/10.1006/expr.1998.4301

    • PubMed
    • Google Scholar

Author details

1. Gabriela C Olivera

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Emily C Ross

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

3. Christiane Peuckert

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Data curation, Formal analysis, Methodology, Validation, Visualization

   Competing interests

   No competing interests declared

4. Antonio Barragan

   Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   antonio.barragan@su.se

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7746-9964

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