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

Abstract

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.

The Ser/Thr Kinase MAP4K4 Controls Pro-Metastatic Cell Functions

“…Since disturbed morphodynamics and cell migration control are intimately associated with the cancerous phenotype, studies in cancer cell lines soon confirmed the promigratory function of MAP4K4 [3,62]. MAP4K4 implications in migration control in the context of cell transformation was also shown in macrophages infected with the protozoan parasite Theileria annulata, where parasite-induced TNFα secretion by the host cell activates MAP4K4 in an autocrine manner, to drive migration and invasion of the host cell [11]. In an inflammatory context as well, MAP4K4 promotes Kaposi Herpes virus-infected cell migration by controlling the expression of genes responsible for motility and invasiveness such as COX-2, MMP-7 and MMP-13.…”

“…These processes include insulin sensitivity [8], systemic inflammation [9], pathogen-dependent oncogenic progression [10,11] and vascular inflammation and atherosclerosis [12]. In addition to the TNFα-receptor, growth factors (GF) such as PDGF or EGF activate MAP4K4 through receptor tyrosine kinases (RTKs) [2] and trigger the phosphorylation and activation of the MAP4K4 substrates sodiumproton exchanger [1] (NHE1) [13], ezrin, radixin, moesin (ERM) family proteins [14] and actin-related protein [2] (Arp2) [15].…”

“…Mechanistically, the identification of C. elegans integrin as an interaction partner of MAP4K4 during commissural axon navigation [16] hinted towards functional implication of MAP4K4 in cell adhesion and migration control. The ezrin, radixin, moesin ERM family were then identified as MAP4K4 substrates and interactors, which mediate lamellipodium extension in response to growth factor (GF) stimulation in breast adenocarcinoma cells [14] and in highly motile macrophages transformed by intracellular pathogen Theileria annulata [11]. Interestingly, in C. elegans, MIG-15 and the moesin orthologue ERM-1 function in the same pathway to direct commissural axon migration [44].…”

“…Common to both studies is that MAP4K4 activity triggers the turnover of adhesion complexes. Indeed, depletion of MAP4K4 or its inactivation causes the cells to spread and adhere more tightly [3,11,14]. Acceleration of focal adhesion turnover may thus be one function of MAP4K4 to facilitate cell displacement, in particularly also inside 3D matrix, where focal adhesions, the adhesion associated signaling complexes and their coordinated turnover controls mesenchymal cancer cell migration [65].…”

“…Arp2 phosphorylation by MAP4K4 increased the actin nucleation activity of the Arp2/3 complex in vitro and in EGF-stimulated cells, demonstrating that MAP4K4 can couple growth factor (GF) signaling to F-actin polymerization [15]. MAP4K4 accumulates at the leading edge of cells migrating on/in either 2D or 3D matrix [3,11,14,49,79]. Thus, MAP4K4-driven F-actin polymerization at the leading edge combined with its activity towards focal adhesions make it a prime candidate for GF control of the membrane protrusions at the leading edge of cells that drive invasive cancer cell motility.…”