Intracellular Theileria annulata Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva) or Tropical Theileriosis (T. annulata). These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell's dissemination capabilities.


Introduction
Theileria annulata is an apicomplexan, intracellular parasite that predominately infects macrophages in vivo. It causes the severe leukoproliferative disorder Tropical Theileriosis in ruminants in northern Africa, the Middle East and Asia where its Hyalomma tick vector is endemic. It is closely related to T. parva, which is transmitted by the tick Rhipicephalus appendiculatus and predominately infects T cells to cause East Coast Fever. Hallmark of infections with T. annulata or T. parva is a host cell transformation process that results in immortalization and permanent proliferation of the infected cell population and -through paracrine stimulation -also of non-infected leukocytes [1].
Theileria-infected cells can be studied in vitro and used as a reversible model of oncogenic transformation because the parasite can be specifically eliminated by parasitocidic treatment with the drug Buparvaquone 720c (BW720c); hence transformationdependent alterations can be determined and pathways that promote these alterations identified [2][3][4]. A series of in vitro and in vivo studies showed that Theileria triggers host cell motile and invasive behavior to facilitate parasite dispersion in the host animal, which is reminiscent of metastatic tumor cell dissemination [5][6][7][8]. TGFß was recently found to trigger a parasitedependent invasive motility program in the host cell through the activation of Rho kinase ROCK [3], analogous to TGFßdependent invasive migration of cancer cells [9]. Earlier studies showed that T. annulata infected mononuclear host cells disseminate as cytokine secreting cells throughout the body in vivo [6]. Cytokines expressed include the pro-inflammatory cytokines IL1ß, IL-6 and TNFa [10,11]; latter was also linked to the maintenance of infected cell proliferation in T. parva-infected B cells through NF-kB activation [12]. TNFa originally identified as a mediator of inflammatory responses with cytotoxic functions, was recognized more recently as a potential inducer of cancer progression by promoting growth and metastatic dissemination of cancer cells in autocrine and paracrine manner [13]. The capability of TNFa to promote dissemination of epithelial tumor cells has been recognized long ago [14]. However, how the underlying molecular mechanisms such as altered integrin av b3 expression via IkB induction [15] or FAN (factor associated with neutral sphingomyelinase activity)-induced actin reorganization via Cdc42 [16,17] affect invasive motility is only poorly understood.
We recently found that host MAP4K4 accumulated at the leading edge of polarized, matrix-invading T. annulata infected host cells [40]. Since MAP4K4 controls inflammatory signaling pathways downstream of TNFa and cancer cell motility, we investigated MAP4K4 functions in macrophages infected with T. annulata, which display a parasite-dependent chronic increase in TNFa expression. We determined the relevance of TNFa signaling for host cell migration and revealed MAP4K4 functions in controlling parasite-dependent actin dynamics regulation and invasive cell motility.

Results
TNFa promotes host cell motility and invasiveness in T.

annulata-infected cells
Previous studies demonstrated a global increase of TNFa in animals parasitized with Theileria [11] and increased TNFa expression in infected macrophages derived both from susceptible and resistant animals [10]. Therefore, we quantified TNFa expression in the three different T. annulata-infected macrophage cell lines TaC12, Thei and TaH12810 by ELISA and qRT-PCR (Fig. 1A). BW720c treatment eliminates parasite within 48 hours and results in populations of parasite-free, viable cells of isogenic background (not shown). In these ''cured'' populations, TNFa secretion was significantly reduced in the Thei and TaH12810 lines and below the ELISA kit's detection limit in the TaC12 line (Fig. 1B). Consistently, TNFa mRNA levels in these cell lines were reduced by 60% to 80% parasite elimination compared to untreated control (Fig. 1C). We observed a similar reduction of TNFa expression after pharmacologcial inhibition of NF-kB (not shown), indicating that permanent induction of NF-kB observed in Theileria-infected cells [2,41] is necessary for increased TNFa expression. To determine whether TNFa induced motile cell behavior, we treated the adherent TaC12 or Thei cells with recombinant TNFa, recorded individual cell movements by time-lapse video microscopy and tracked cell trajectories (paths). We plotted cell paths (Fig. S1A) and measured their lengths (Fig. 1D). We found that TNFa treatment significantly increased path lengths, which proportionally reflect cell speeds. Interestingly, TNFa treatment did not affect directionality of migration as neither the directional migration index (distance/path length, DMI, Fig. S1B), nor the average angular deviations of the axis nucleus-leading edge (Fig. S1C) changed. Hence we concluded that increased cell displacement after TNFa treatment is the result of increased speed and not of increased directional persistence of migration. We then compared matrigel invasion of control or TNFastimulated TaC12 cells using matrigel-coated Boyden chambers ( Fig.  S1D and 1E). We found that TNFa-stimulated cells had a significantly higher capability to transmigrate, indicating that TNFa promoted invasive capabilities of T. annulata-infected cells.
Lamellipodia are the driving structures for infected cell movements in 2D [4]. Therefore, we compared actin dynamics in lamellipodia of control or TNFa-treated cells by fluorescence live cell imaging using lifeact-EGFP (LA-EGFP). We detected no quantifiable differences in actin dynamics between untreated and TNFa-treated cells (not shown). However, when we determined the size of lamellipodia relative to the size of the whole cell (Fig. 1F, left), we found that TNFa promoted a significant increase in lamellipodium size in TaC12 and Thei cells (Fig. 1F, right), while overall cell size was not affected (S1E). More importantly, cells embedded in matrigel developed invasive protrusions (Fig. 1F, ''3D'') reminiscent of those observed in the recently isolated and virulent strain TaH12810 [40]. To determine whether TNFa treatment only stabilizes lamellipodia or also triggers their assembly, we investigated the impact of TNFa stimulation on lamellipodium formation in BW720c-treated cells, which lack lamellipodia. Comparable to the Thei macrophages investigated in our previous study [4] and Fig. 1G, right, the number of TaC12 cells with a single lamellipodium was drastically reduced after parasite elimination (Fig. 1G). TNFa stimulation of cured TaC12 or Thei cells rescued single lamellipodia assembly, strongly indicating that TNFa meditates motility and invasiveness through the control of lamellipodium assembly and stabilization.
Taken together, we found that TNFa production and secretion is parasite-dependent and that TNFa promotes motility and invasiveness of T. annulata-infected host cells, likely due to the capability of TNFa to enhance F-actin-rich leading edge protrusions.

Secreted TNFa is necessary for efficient motility and invasiveness of infected cells
To determine whether endogenous TNFa is sufficient to promote motility and invasiveness, we silenced TNFa expression in TaC12

Author Summary
The protozoan parasite Theileria annulata causes the often fatal leukoproliferative disorder Tropical Theileriosis in their ruminant host animals, which is the result of widespread dissemination and proliferation of cytokine secreting, parasite-infected cells. This host cell behavior is induced by and dependent on the intracellular presence of the parasite and is reminiscent of metastatic dissemination of human cancer cells. We investigated how the intracellular parasite modulates cell motility and invasiveness, to better understand the pathogenesis of Tropical Theileriosis and to reveal conserved mechanisms of eukaryotic cell motility regulation. We found that the parasite drives host cell motility and invasiveness through the induction and activation of the host cell protein MAP4K4. We show that MAP4K4 induction is driven by the inflammatory cytokine TNFa and causes dynamic changes in the cytoskeleton of the host cell that facilitate cell motility. Thus, our findings reveal how the intracellular Theileria parasite can influence morphology and behavior of its host cell in a way that suits its propagation and highlight a novel function of chronic TNFa production for the pathogenesis of Tropical Theileriosis. Furthermore, our study revealed a novel aspect of inflammatory cytokine action, namely cell mobilization through the induction of the evolutionary conserved protein kinase MAP4K4. cells by two different siRNAs. We effectively reduced the quantity of soluble TNFa protein in the culture supernatants ( Fig. 2A), although TNFa mRNA was only moderately reduced (Fig. S2A). Under conditions of reduced soluble TNFa, motility (path lengths) of infected Tac12 and Thei cells was reduced by 60% ( Fig. 2B & C) and lamellipodium formation impaired (Fig. S2D). Silencing endogenous TNFa had no effect on directionality of migration (Fig. S2). We next tested whether reduced endogenous TNFa would reduce matrigel invasion, conversely to increased invasiveness after ectopic addition of TNFa. Indeed, decreasing TNFa significantly reduced matrigel invasive and transmigratory capability of TaC12 cells (Fig. 2D &E). Thus, secreted TNFa promotes infected cell motility by increasing the speed of cell migration and by increasing the capability of cells to penetrate stiff matrices. We conclude that T. annulata-induced TNFa alters motile cell behavior in a way that facilitates dissemination of infected cells, while the source of TNFa can be both autocrine and paracrine.

MAP4K4 is constitutively induced and activated in infected cells
Given the pro-migratory effects of TNFa on T. annulata-infected macrophages, we searched for a downstream effector controlling actin dynamics and cell motility. MAP4K4 is TNFa-induced kinase, signaling through the JNK pathway [19,24] and contributing to migration and metastasis of cancer cells [27][28][29]. In T. annulata-infected macrophages, we detected MAP4K4 in the nucleus and in leading edge lamellipodia (Fig. 3A). We next determined whether MAP4K4 protein expression is parasitedependent by comparing MAP4K4 protein abundance in parasitized and BW720c drug-cured cells by immuno blotting (IB). Fig. 3B upper shows a representative IB with antibodies against MAP4K4, the ezrin, radixin, moesin (ERM) proteins, the Src kinase Hck and tubulin. Average expression levels indicated a moderate (50%) reduction of MAP4K4 protein abundance in cured cells (Fig. 3B, lower). We confirmed moderate MAP4K4 down regulation after parasite elimination in the Thei (Fig. S3A) and TaH12810 (Fig. S3B) lines. Parasite elimination also reduced autophosphorylation capabilities of immunoprecipitated MAP4K4 (Fig. 3C) and its activity towards the substrate MBP in vitro (Fig.  S3C), indicating that chronic infection of macrophages by T. annulata increases MAP4K4 expression and activity. To test whether a secreted host cell factor promoted MAP4K4 expression, we treated TaC12 cells with conditioned medium. 24 h later, we monitored alterations in MAP4K4 expression by IB. Interestingly, conditioned medium promoted increased MAP4K4 expression with maximal effects observed at 50% conditioning (Fig. 3D), suggesting that MAP4K4 expression is induced in an autocrine manner by a parasite-dependent, host cell secreted factor.

MAP4K4 promotes motility and invasiveness of infected cells
MAP4K4 was identified as a pro-migratory kinase in carcinoma cells [27]. Therefore, we measured whether siRNA-mediated silencing of MAP4K4 would impair infected cell motility. We tested three different MAP4K4-specific siRNAs, from which two (siMAP4K4_1 and siMAP4K4_2) effectively reduced MAP4K4 expression, both at the mRNA and protein levels (Fig. 3E). We quantified path lengths and directionalities of siControl and siMAP4K4 cells by live-cell imaging. Silencing MAP4K4 significantly reduced path length over time (speed) (Fig. 3F), while it did not affect directionality of migration ( Fig. S4A & B). Consistently, EGFP-fused wild-type (wt, movies S1 & S2) but not kinase dead (k/d, movies S3 & S4) MAP4K4 promoted a motile phenotype when ectopically expressed in TaC12 cells. We next compared the capability of siMAP4K4 cells to cross matrigel-coated Boyden chambers and found that matrigel invasion was significantly reduced when MAP4K4 was depleted (not shown). Importantly, MAP4K4 silencing also blocked TNFa-induced F-actin-rich cell protrusions (

TNFa induces MAP4K4 expression
To determine whether TNFa induced MAP4K4 expression, we examined alterations in MAP4K4 expression by IB in TaC12 cells treated with recombinant TNFa. We found that TNFa upregulated MAP4K4 proteins in a dose dependent manner, with a 3.5 fold increase at 25 ng/ml concentration (Fig. 4A). We also tested whether the two unrelated cytokines HGF, which could induce MAP4K4 in a heterologous system [42] and GM-CSF, which promotes proliferation of Theileria infected cells [43] affected MAP4K4 expression. Neither HGF nor GM-CSF significantly increased MAP4K4 expression within 24 hours of treatment (not shown). TNFa activates the JNK signaling pathway [20], which is constitutively activated in different Theileria-infected cell lines [44][45][46]. Consistently, phosphorylation of JNK on Thr183 and Tyr185 and of its downstream substrate ATF2 on Thr71 in TaC12 cells is increased in a time-dependent manner after TNFa treatment (Fig. 4B). Pre-treatment with the JNK inhibitor SP600125 prevented TNFa-induced JNK phosphorylation and activation ATF2 (Fig. 4B, right). We next silenced MAP4K4 before TNFa treatment and found that MAP4K4 depletion markedly reduced TNFa-induced activation of the JNK substrate ATF2 (Fig. 4C), indicating MAP4K4 functional relevance in TNFa-dependent JNK pathway activation in T. annulata-infected cells. Interestingly, JNK signaling was needed for TNFa-induced MAP4K4 expression because the stimulatory effect of TNFa on MAP4K4 expression was inhibited when JNK activity was pharmacologically blocked (Fig. 4D).
Thus, TNFa contributes to JNK signaling in infected cells and promotes MAP4K4 expression most likely via JNK-mediated ATF2 activation [19], while MAP4K4 in turn is needed for TNFa-induced JNK activation. Hence, the MAP4K4-JNK signaling module is active in infected cells and integrates TNFa signaling into MAP4K4 induction in a positive feedback type mechanism.

MAP4K4 expression is regulated at transcriptional and post-transcriptional levels
Interestingly, qRT-PCR analysis revealed strikingly higher MAP4K4 mRNA levels in cured cells (Fig. 5A). Therefore, we hypothesized that MAP4K4 expression could be transcriptionally up-regulated after parasite elimination. The only transcription factor described so far besides ATF2 [19] regulating MAP4K4 is p53 [47]. T. annulata was recently shown to sequester host cell p53 in infected cells [48]. Indeed, in TaC12 cells, p53 gradually translocated from the parasite surface to the host cell nucleus after BW720c treatment (Fig. 5B). Therefore, we blocked p53 function in cured cells with Pifithrin [49] and quantified MAP4K4 mRNA expression by qRT-PCR. We found that BW720c-induced MAP4K4 was completely abrogated by Pifithrin (Fig. 5C), suggesting that p53 promotes increased MAP4K4 mRNA induction in drug-cured cells. Conversely, when we triggered p53 function in parasitized cells, either by induction (etoposide, Fig. 5D) or stabilization (Nutlin-3, Fig. 5E), we observed a timedependent increase in MAP4K4 expression. Both compounds led to the accumulation of p53 in the nucleus of infected cells, indicative for p53 activation (Fig. S5A). Moderate proteasomemediated MAP4K4 degradation after BW720c treatment (Fig.  S5B) cannot fully account for reduced MAP4K4 protein in the presence of high levels of MAP4K4 mRNA. We alternatively hypothesized that MAP4K4 mRNA is translationally blocked by miRNA binding to the 39 UTR of the MAP4K4 mRNA. Using TaC12 cells stably expressing luciferase fused to MAP4K4 39UTR or to GAPDH 39UTR as control, we found that parasite elimination significantly and specifically reduced luciferase-MAP4K4 39UTR expression, suggesting that the presence of T. annulata in the host macrophage repressed miRNA targeting of the 39UTR of MAP4K4 (Fig. 5F). Together, our data show that increased MAP4K4 mRNA after parasite elimination requires functional host cell p53 induction and indicate miRNA-mediated translational repression of newly synthesized MAP4K4 mRNA.

Regulation of lamellipodia dynamics by MAP4K4 involves ERM protein activation
Approximately 70-80% of TaC12 cells harbor one to three large, F-actin-rich lamellipodia (Fig. 1F, 3A & 6A). Silencing MAP4K4 reduced this number to 47 and 46% for siMAP4K4_1 and siMAP4K4_2, respectively (Fig. 6A), indicating MAP4K4 implication in lamellipodium assembly. The membrane-F-actin cross-linker proteins of the ezrin, radixin and moesin (ERM) family accumulate in persistent lamellipodia of T. annulata-infected macrophages [4] whereas MAP4K4 phosphorylates ERM proteins on the regulatory C-terminal threonine residue to control lamellipodia dynamics [50]. Therefore, we tested by IFA as to whether MAP4K4 could control ERM protein accumulation in lamellipodia of T. annulata-infected cells. In control siRNA transfected cells, ERM proteins accumulate throughout lamellipodia, while activated (phosphorylated) ERM (pERM) proteins localize more distally towards the leading edge (Fig. 6B), where also MAP4K4 localizes (Figs. 3A & 6A). MAP4K4 depletion markedly reduced lamellipodia formation and, consequently, also ERM protein accumulation in lamellipodia (Fig. 6C). Reduced ERM accumulation under siMAP4K4-treated conditions was also evident in cells that displayed F-actin-rich lamellipodia (Fig. 6C, compare c and d). In MAP4K4 depleted cells, pERM was detectable in lateral and basal filamentous protrusions when no lamellipodia were present, possibly representing MAP4K4-independent ERM protein phosphorylation (Fig. S6) also detected by IB (Fig. 7A). Interestingly, TNFa treatment led to a marked global increase in C-terminal ERM phosphorylation (Fig. 7A). In MAP4K4 depleted cells, TNFa-induced ERM phosphorylation was no longer evident. This indicated that it was MAP4K4 that mediated ERM protein phosphorylation downstream of TNFa, while another kinase must contribute to the maintenance of the global pERM levels in unstimulated cells, because non TNFainduced pERM was not reduced in MAP4K4-depleted cells. This observation is consistent with the findings of our IFA analyses (Fig.  S6), where lammellipodia-localized but not global pERM was altered. In lamellipodia of TNFa-treated cells, we observed focal accumulations of pERM proteins (Fig. 7B). We compared fluorescent intensities of pERM in lamellipodia of unstimulated and TNFa-stimulated cells and quantified a moderate but significant 24% increased in pERM in stimulated cells (Fig. 7C). However, TNFa treatment did not rescue lamellipodia formation in cells where MAP4K4 was previously silenced (Fig. 6a & 7D), corroborating the notion that TNFa is upstream of MAP4K4 regulation of lamellipodia dynamics. Together, our data show that TNFa induces ERM phosphorylation on the regulatory C-terminal threonine in a MAP4K4-dependent manner and that this ERM phosphorylation likely occurs in lamellipodia.

Discussion
In this manuscript we established TNFa-dependent induction of host MAP4K4 as a novel mechanism triggered by the intracellular presence of the parasite T. annulata to promote dissemination of its host cell. We identified MAP4K4 as a critical intermediate that bifurcates TNFa signaling to JNK pathway activation on one hand and to the activation of the ERM family of cytoskeleton regulatory proteins and actin dynamics on the other hand. JNK pathway activation ensures MAP4K4 expression, while ERM activation spatio-temporally correlated with persistent lamellipodia formation and invasive cell motility (schema Fig. S7). We propose MAP4K4 as a novel effector kinase linking TNFa signals to invasive cell motility regulation under conditions of chronic exposure to TNFa, such as during pathogen infections, in inflammatory disorders and in cancerous lesions.
Several groups previously reported chronic TNFa induction in Theileria-infected cells. However, since TNFa expression levels were comparable in ex vivo cultures derived from T. annulatainfected cattle breads of different disease susceptibilities, its impact as host virulence factor was not further investigated [10]. Guergnon et al. linked TNFa expression to NF-kB induction and infected cell proliferation [12], consistent with studies in other systems demonstrating TNFa impact on pro-and anti-apoptotic signaling and its role in inflammation regulation [13]. Here, by characterizing its role in promoting cell motility, we revealed an additional function of TNFa that is largely independent of its role in proliferation/survival regulation. By demonstrating a mechanistic link of TNFa signaling to the established motility regulator MAP4K4 [27][28][29][30][31], we provide an explanation for how TNFa expression contributes to Tropical Theileriosis pathogenesis and how TNFa production could be linked with the progression of tumors to invasive cancers [13]. We found that a secreted factor controls MAP4K4 expression because conditioned medium derived of infected cells promoted the expression of MAP4K4. This effect was phenocopied by the addition of recombinant TNFa to unconditioned, fresh medium. Optimal MAP4K4 induction was achieved at 50% conditioning (rather than at 100%), possibly either due to nutrient starvation in media with a higher ratio of conditioned medium or due to an MAP4K4 inhibitory factor secreted by cells grown to confluence. Unlike TGFb, who's expression is increased in infected cells derived from susceptible host animal [3], TNFa expression was found to be similar in cattle breeds with different disease susceptibilities [51]. Hence, TNFa and TGFb action could synergize to promote host cell virulence, whereby TNFa enables primary motile properties that are further enhanced after exposure to TGFb. Importantly, TGFb controls the expression of additional pro-migratory factors including hepatocyte growth factor (HGF) [3], which can promote motility of Theileria-infected cells without affecting MAP4K4 expression (Ma and Baumgartner, unpublished observation). Thus, Theileria-infected cells produce a mixture of factors including TGFb, HGF and TNFa that contribute individually and collectively to cell motility, whereby TGFb and TNFa are coupled directly to cell motility regulation through Rho-kinase ROCK [3] and MAP4K4 (herein), respectively. Right: IB analysis of effect of JNK inhibitor SP600125 (10 ng/ml, added 2 h before TNFa treatment) on TNFa-induced JNK pathway activation. Bar diagram: Means and SDs of pJNK and pATF2 after TNFa stimulation (normalized to total JNK and total ATF2 proteins, respectively). C) Time course IB analysis of lysates of cells treated with 25 ng/ml TNFa using antibodies against proteins indicated. Cells were stimulated 24 h after transfection with siControl or siMAP4K4_1 or siMAP4K4_2. Bar diagram below shows quantification of pATF2 bands (normalized to total ATF2 protein). D) IB analysis of lysates from cells treated for 24 h2/+25 ng/ml TNFa and 2/+10 ng/ml SP600125, using anti-MAP4K4 or anti-tubulin antibodies. Bar diagram below shows quantification of MAP4K4 bands (means 2/+ SDs, normalized to tubulin). doi:10.1371/journal.ppat.1004003.g004 We propose the promigratory effect of MAP4K4 to be due to its capability to affect actin dynamics in cellular protrusions by direct C-terminal phosphorylation of ERM family proteins [50], and possibly also by alternative mechanisms including direct phosphorylation of actin modulatory proteins [52]. Indeed, ERM protein activation is associated with parasite-dependent host cell polarization [40,53], whereas it is also considered an important event during cancer metastasis [54,55]. Although it is still not entirely clear how similar the processes are that drive invasiveness of Theileria-infected cells and cancer cells, polar activation of ERM proteins is a mechanism likely generally conserved in invading cells. Several C-terminal ERM kinases have been proposed as ERM activators (reviewed in [56]) to modulate the cortical actin cytoskeleton and morphodynamic processes [57]. Depletion of MAP4K4 by siRNA completely abrogates TNFa-induced ERM phosphorylation, suggesting that it is mainly MAP4K4 that mediates C-terminal ERM phosphorylation downstream of TNFa in T. annulata-infected cells. Using pharmacological approaches, ERM phosphorylation downstream of TNFa was also previously shown and linked to p38 MAPK and PKCs in endothelial cells [58] or p38 MAPK and Rock activities in fibroblast-like synoviocytes [59]. However, both studies showed ERM phosphorylation kinetics that peaked after one hour and contrast our finding of rapid (within 5 min) ERM phosphorylation, suggesting that immediate ERM phosphorylation by MAP4K4 is direct. Interestingly, a recent study revealed phosphorylation of the ERM protein moesin by JNK for podosome rosette formation in Srctransformed fibroblasts [60]. It is conceivable that an analogous JNK-mediated phosphorylation of ERM proteins may be active as well for parasite-dependent podosome formation in Theileriatransformed cells, which requires Src kinase activity too [4], possibly causing residual ERM phosphorylation in MAP4K4depleted cells. Consistent with the notion of selective, spatially restricted activity of JNK and MAP4K4 towards ERM proteins, we observed no co-localization of MAP4K4 with podosomes in infected cells. ERM activation by MAP4K4 likely structurally stabilizes the interaction of newly polymerized F-actin filaments with membrane proteins, to promote lamellipodia in 2D and invasive protrusions in 3D and to facilitate assembly of signaling complexes. ERM proteins can also act as protein kinase A anchoring proteins to affect cAMP-induced signaling pathways [61], suggesting that TNFa-induced ERM phosphorylation could also affect PKA signaling active in T. annulata-infected cells [62]. MAP4K4 expression at the mRNA level increased after parasite elimination while MAP4K4 protein decreased. We explain this conundrum with two unrelated but parasite-dependent mechanisms. First, using either specific inhibitors or activators of p53, we concluded that p53 released from the parasite surface -where it is sequestered when the parasite is intact [48] -can transcriptionally induce MAP4K4. The use of a proteasome inhibitor under these conditions did not indicate that the consequently increased amounts of MAP4K4 protein is diminished by proteasomal degradation; rather our data indicate miRNA-dependent targeting of the MAP4K4 39UTR to limit MAP4K4 protein expression.
Thus, MAP4K4 protein abundance is regulated by JNK-ATF2 signaling in parasitized cells and after parasite elimination, by p53 combined with translational repression by miRNA. Indeed, parasite-dependent regulation of host cell miRNA expression has just recently been demonstrated [63]. miRNA targeting of MAP4K4 could be a mechanism evolved to protect cells from overabundance of this potentially oncogenic kinase.
Together, we highlight the potential functional significance of TNFa signaling in Tropical Theileriosis, and we revealed a pathogen dependent-mechanism controlling oncogene expression and function to promote invasive motility of its host cell. Analogous to Theileria-induced host cell transformation, MAP4K4 signaling might be relevant in driving cancer cell dissemination in tumors, where increased levels of anti-cancer therapy-induced TNFa has been noted in the surrounding stroma [64].

Matrigel 3D cultures
Cells were suspended in RPMI-1640 medium and mixed with growth factor reduced Matrigel (354230, BD BioScience) at a ratio of 1:9. 10 ml of the cell-matrigel suspension were transfer per well into 15 well m-Slide (81506, Ibidi). After polymerization of the matrigel, the wells were filled with 50 ml medium (with or without treatment). Cells were fixed by 4% PFA after 24 hours in culture.

Matrigel invasion assay
For matrigel invasion assays, matrigel-coated Boyden chambers (BD 354480) were used. 25'000 cells were suspended in RPMI (0.5% FBS) and seeded on the upper side of the matrigel-coated membrane; the lower side of the membrane was submerged in complete medium or 0.5% FBS medium supplemented with 25 ng/ml TNFa. After incubation for 24 hours at 37uC, transmigrated cells passing through the membrane were fixed with 4% PFA, stained with 0.05% crystal violet, and viewed and counted using a bright-field microscope.

Single-cell motility assay
Cells were seeded on 8 well chamber slides (80826, ibidi) at 40% confluency in assay medium with or without TNFa. Time-lapse movies were acquired on an automated Leica LX microscope equipped with a Hamamatsu EM-CCD camera in differential interference contrast (DIC) bright field modus using a 106dry NA 0.3 objective lens. Cells were maintained at 37uC in a humidified atmosphere containing 5% CO 2 . Cell speed was determined by manually tracking the cells every 5 minutes for 6-12 hours using ImageJ software (NIH image J software).
Directionality of migration index (DMI) in a given time equals the quotient of the rate of displacement divided by the length of the corresponding path, yielding a number between 0 and 1.
Average angular deviations in radians of the axis nucleusleading edge were determined by assessing the angular deviations of one time point from the corresponding axis of one time point earlier.

ELISA
TNFa concentrations in cell culture supernatants were measured by ELISA (Bethyl, E11-807) according to manufactur-er's instructions. Supernatant samples for TNFa ELISA were collected from cells 24 h after passaging into fresh medium. BW720c-treated cells were treated for 48 h and then re-seeded in fresh medium. si-RNA-transfected cells were re-seeded in fresh medium 24 h after transfection. For both treatments, samples for ELISA were taken 24 h after re-seeding and compared to compared to samples derived from the same number of control cells.

Actin dynamics
TaC12 cells were infected with pLENTI-LA-EGFP. Actin dynamics were then monitored by live-cell microscopy using a Leica SP5 confocal microscope with temperature and climate control. Images were acquired using a Hamamatsu EM-CCD camera and assembled and analyzed using Imaris software.

RNA extraction and Real-Time Quantitative PCR
Total RNA was isolated (kit 74106, QIAGEN) and reverse transcribed (Applied Biosystems) using oligodT primers according to manufactures' instructions. qPCR reactions (Applied Biosystems) were performed on an ABI 7900HT apparatus. Primers are listed in paragraph ''primer and siRNA sequences''. All quantifications were normalized to control endogenous GAPDH. Relative changes in gene expression were quantified using the 2-DDC T methods.

Plasmid transfection
Plasmids were transfected using the Amaxa nucleofection protocol with SF solution and program DS103. 0.5610 6 cells were used per transfection with 1.5 mg plasmid DNA in 6.5 ml SF solution and 3.6 ml supplement.

Luciferase 39UTR assays
pLenti-luc-hsGAPDH-39UTR and pLenti-luc-hsMAP4K4-39UTR (Applied Biological Materials, Richmond, Canada) constructs were used to generate lentiviruses. The majority of miRNA target sites in the human MAP4K4 39UTR predicted to bind specific miRNAs using TargetScan software are also conserved in the bovine MAP4K4 39UTR (see supporting table S1). Stable TaC12 cell lines that express luciferase under the respective 39UTR control were generated by puromycin selection. Variation in transgene abundance between GAPDH-39UTR and MAP4K4 39UTR in stable lines was determined using qRT-PCR with luciferase-specific primers and used to normalize luciferase measurements. Luciferase activity was determined according to manufacturer's (Promega) instructions.

Statistical analysis
Statistical analysis and significance tests were performed using Prism5 software. Data are calculated as means 6 SDs and expressed either as means of absolute values or as means of changes relative to control treatments. P values were determined using two-tailed paired or unpaired students T-test. Paired T-tests were performed when changes in a pair before-after treatment were compared. Normal distribution of sample values (n$30) was confirmed when multiple individual measurements were compared (cell tracking, relative lamellipodia sizes, directionality indices). Choice of cells and fields was always random to minimize bias. Box plots show 25th percentile, median and 75th percentile; whiskers show minimal to maximal distributions. At least 3 independent experiments were performed. P values of ,0.05 were considered statistically significant. T-tests: * = p,0.05; ** = p,0.001 and *** = p,0.0001.