Intracellular <i>Theileria annulata</i> Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

<div><p>The intracellular, protozoan <i>Theileria</i> 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 <i>in vitro</i> and their metastatic dissemination in the animal, which causes East Coast Fever (<i>T. parva</i>) or Tropical Theileriosis (<i>T. annulata</i>). 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 <i>T. annulata</i>-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 <i>Theileria</i> parasite to increase its host cell's dissemination capabilities.</p></div

TNFα promotes increased MAP4K4 protein expression through the JNK pathway.

<p><b>A</b>) IB analysis of cells treated for 24 h with increasing concentrations of TNFα using antibodies against MAP4K4 or tubulin; Below: quantification of integrated densities of bands (means −/+ SDs, normalized to tubulin). <b>B</b>) Time course IB analysis of lysates of cells treated with 25 ng/ml TNFα using antibodies against proteins indicated. Right: IB analysis of effect of JNK inhibitor SP600125 (10 ng/ml, added 2 h before TNFα treatment) on TNFα-induced JNK pathway activation. Bar diagram: Means and SDs of pJNK and pATF2 after TNFα stimulation (normalized to total JNK and total ATF2 proteins, respectively). <b>C</b>) Time course IB analysis of lysates of cells treated with 25 ng/ml TNFα 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). <b>D</b>) IB analysis of lysates from cells treated for 24 h−/+25 ng/ml TNFα and −/+10 ng/ml SP600125, using anti-MAP4K4 or anti-tubulin antibodies. Bar diagram below shows quantification of MAP4K4 bands (means −/+ SDs, normalized to tubulin).</p

MAP4K4 promotes host cell motility and TNFα-induced invasiveness.

<p><b>A</b>) Confocal IFA of intracellular localization of MAP4K4 in parasitized (upper) and BW720c-cured (lower) TaC12 cells. Red: actin, green: MAP4K4, blue: TaSP. Right: magnification of TaC12 lamellipodium showing accumulation of MAP4K4 at leading edge. Schema shows localization of MAP4K4 at leading edge of cell. <b>B</b>) IB analysis of untreated and BW720c-treated TaC12 cells using anti-MAP4K4, anti-ERM, anti-Hck and anti-tubulin antibodies. Bar diagram below shows mean protein abundances −/+ SDs. in control and BW720c-treated TaC12 cells. <b>C</b>) <i>In vitro</i> kinase assay comparing MAP4K4 immunoprecipitated either from infected or cured TaC12 cells. Upper: IB of immunoprecipitated MAP4K4, lower: autorad of total protein phosphorylation activities immunoprecipitated. <b>D</b>) Upper: IB analysis with anti-MAP4K4 and anti-tubulin antibodies using lysates of TaC12 cells previously incubated with increasing concentrations of conditioned medium. Lower: Mean integrated densities of MAP4K4 bands (−/+ SDs, normalized to tubulin) <b>E</b>) Quantification of MAP4K4 depletion by qRT-PCR using primers p81 and p79 (upper, see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat-1004003-g005" target="_blank">Fig. 5A</a>) or IB (lower) 24 h after siControl or siMAP4K4 transfection. <b>F</b>) 12 h single cell migration analysis of cells transfected with either siControl or siMAP4K4_1 or siMAP4K4_2. Box plot show means of path lengths (n = 34, 38, 44). <b>G</b>) Matrigel invasion assay: Bright field microscopy images (100× magnification) of transmigrated siControl or siMAP4K4_1 or siMAP4K4_2 transfected cells after 24 h in the presence of 25 ng/ml TNFα. <b>H</b>) Quantification of images shown in G expressed as relative transmigration compared to siControl and SDs. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat.1004003.s003" target="_blank">Figures S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat.1004003.s004" target="_blank">S4</a>.</p

Host cell-secreted TNFα is required for parasite-induced motility.

<p><b>A</b>) Quantification of TNFα depletion in TaC12 and Thei culture supernatants by ELISA, 48 h after siControl or siTNFα transfections. Red line: detection limit (0.078 ng/ml). <b>B</b>) Paths of individual siControl or siTNFα-transfected TaC12 cells migrating for 8 h. <b>C</b>) Quantifications of path lengths of control and TNFα-depleted TaC12 and Thei cells. Box plots with means of path lengths are shown (TaC12 n = 88, 95, 105 and Thei n = 35, 35, 35) <b>D</b>) Matrigel invasion assay: Bright field microscopy images (100× magnification) of either siControl or siTNFα_1 or siTNFα_2 transfected cells after transmigration. <b>E</b>) Quantification of secreted TNFα dependent transmigration in matrigel assay after 24 h. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat.1004003.s002" target="_blank">Figure S2</a>.</p

The ERM kinase MAP4K4 localizes to leading and trailing edges of migrating TaH12810 cells.

<p><b>A</b>) IF microscopy analysis of ERM and MAP4K4 protein localization in cells migrating in matrigel using anti-ERM and anti-MAP4K4 antibodies. Magnifications are 4x of boxed area and highlight MAP4K4 accumulation in membrane blebs. Arrows indicate direction of migration. <b>B</b>) Time-lapse microscopy of TaH12810 cells expressing either YFP-ezrin (movies S11 & S13) or EGFP-MAP4K4 (movies S9 & S10). LA-mCherry was used to visualize leading edge actin dynamics (movie S12). Cells migrate in matrigel either in saltatory (upper) or tunneling (lower) mode (from left to right: grey scale, GFP or YFP fluorescence, overlay). Dotted line indicates cavity boundary, stars indicate leading edge of cells.</p

Modes of motility of

<div><p><i>T</i><i>. annulata</i>-<b>infected </b><b>macrophages </b><b>embed </b><b>in </b><b>matrigel</b>. </p> <p>(A) Motility of cells embedded in collagen, matrigel or Qgel matrices was recorded for 15 h by live-video microscopy. Vertical scatter dot plots show speed calculated as µm/min (collagen: 55 cells, matrigel: 19 cells, QGel: 10 cells). T-test, *** p < 0.0001. B) Infected cells adopt different modes of motility in matrigel. These include forward movement by tunneling, which requires matrix degradation/engulfment at the leading edge (movie S3 and S5). Alternatively, cells can move in a saltatory mode, where cells squeeze through narrow pores and forward movement oscillates between rapid movements and phases of stagnation (movie S4 and S6). Vertical scatter dot plots show percentage of cells migrating in tunneling (14%) or saltatory (38%) mode. 48% of the cells could not be allocated to one of the two modes (immobile). 147 cells total, T-test, ** p = 0.0014. C) X/Y plots show representative paths of cells migrating in the tunneling (left) or in the saltatory mode (right). Dot plots show representative track lengths of tunneling and saltatory migration in µm. Speed diagrams compare fluctuations in speed development of tunneling and saltatory movements; peak = maximum speed, valley = minimal speed or stagnation.</p></div

ERM proteins localize sequentially to leading edge, the neck zone and the trailing edge.

<p><b>A</b>) If microscopy analysis of ERM protein localization in cells migrating in matrigel using anti-ERM antibodies (red). Leading edge of cells was determined using cortactin localization (green). The location of the trailing edge was confirmed by visualizing host and parasite nuclei (arrows) using hoechst stain. Arrowheads indicate neck zones. <b>B</b>) Quantification of YFP-ezrin fluorescence intensity (leading to trailing edge). Top row shows still images of YFP-fluorescence of time lapsed-image acquisition for a period of 9 min. Bottom row are the corresponding gray-scale images. Middle row shows intensity histograms of YFP-fluorescence along the white line (top row left). Arrows indicate tail, arrowhead leading edge.</p

Membrane blebs evolve along filopodia-like protrusions at leading edge.

<p><b>A</b>) Still images of DIC live-cell microscopy (movie S14). Arrowheads indicate filopodia-like protrusions, arrow neck zone of maximal compression. <b>B</b>) Higher magnification of leading edge of cell shown in A. Arrowhead highlights blebbing protrusion at maximal expansion. <b>C</b>) Quantification of surface area of blebbing protrusion shown in B. <b>D</b>) Confocal microscopy image of TaH128101 cells invading matrigel. Green: F-actin, red: parasite surface (anti-TaSP), blue: DNA. Insets show leading F-actin and host cell nuclear DNA. <b>E</b>) as D but cells were incubated in the presence of either the Src kinase inhibitor PP2 (5 µM) or the Rho kinase inhibitor H-1152 (5 µM). <b>F</b>) model of F-actin distribution and leading edge membrane dynamics of TaH12810 cells invading matrigel.</p

Actin polymerization at invasion front of cells migrating in saltatory mode.

<p><b>A</b>) TaH12810 cells were embedded in matrigel and migration was monitored by time-lapse imaging. Green dots indicate circumference of cavities in migration path, red lines indicate position of pores between holes. <b>B</b>) Time-lapse microscopy of EGFP-actin expressing TaH12810 cell invading matrigel (movies S7 and S8). Top: grey scale images, bottom: EGFP-actin fluorescence. <b>C</b>) 3D reconstruction of confocal microscopy sections of TaH12810 cell migrating in matrigel. Actin cytoskeleton stained with TRITC-phalloidin (red). Arrow indicates actin–rich circle in maximal constriction zone at pore after nuclear translocation (inset shows F-actin in gray scale, 2x magnification). <b>D</b>) As in B but focus on initial phase of matrigel invasion. EGFP-actin fluorescence intensity was measured along line indicated and plotted against line length in µm. Arrows indicate peak fluorescence at cell cortex where protrusion emerges, arrowheads F-actin-rich ring at circular constriction zone, asterisks leading edge F-actin.</p

MAP4K4 promotes lamellipodium formation and enhances ERM accumulation in lamellipodia.

<p><b>A</b>) Confocal IFA of endogenous MAP4K4 (green), F-actin (red) and TaSP (blue) in cells transfected with siControl or siMAP4K4. Bar diagram shows quantification of percent cells displaying a large, single lamellipodium in the absence (black) or presence (white) of 25 ng/ml TNFα. C: siControl; 1: siMAP4K4_1; 2: siMAP4K4_2 <b>B</b>) IFA of total ERM (anti-ERM ab) proteins and ERM proteins phosphorylated on C-terminal threonine (anti-pERM ab) in cells seeded on Fn. <b>a</b> and <b>b</b> show 4× magnifications of boxed areas for ERM (<b>a</b>) and pERM (<b>b</b>) distribution. F-actin is in red, ERM and pERM in green, DNA in blue. <b>C</b>) IFA of ERM localization in siControl or siMAP4K4 transfected cells. IFA as in B, <b>c</b> and <b>d</b> show 4× magnifications of boxed areas for ERM accumulation in lamellipodia of siControl (<b>c</b>) and or siMAP4K4 transfected cells (<b>d</b>). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat.1004003.s006" target="_blank">Figure S6</a>.</p