The level of H2 O2 -type oxidative stress regulates virulence of Theileria-transformed leukocytes

International audienceTheileria annulata infects predominantly macro-phages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macro-phages are aggressively invasive, but this viru-lence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their charac-terization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of mono-cytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α acti-vates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes main-tain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H 2O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels conse-quently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence

The Microtubule-Stabilizing Protein CLASP1 Associates with the Theileria annulata Schizont Surface via Its Kinetochore-Binding Domain

Theileria is an apicomplexan parasite whose presence within the cytoplasm of a leukocyte induces cellular transformation and causes uncontrolled proliferation and clonal expansion of the infected cell. The intracellular schizont utilizes the host cell's own mitotic machinery to ensure its distribution to both daughter cells by associating closely with microtubules (MTs) and incorporating itself within the central spindle. We show that CLASP1, an MT-stabilizing protein that plays important roles in regulating kinetochore-MT attachment and central spindle positioning, is sequestered at the Theileria annulata schizont surface. We used live-cell imaging and immunofluorescence in combination with MT depolymerization assays to demonstrate that CLASP1 binds to the schizont surface in an MT-independent manner throughout the cell cycle and that the recruitment of the related CLASP2 protein to the schizont is MT dependent. By transfecting Theileria-infected cells with a panel of truncation mutants, we found that the kinetochore-binding domain of CLASP1 is necessary and sufficient for parasite localization, revealing that CLASP1 interaction with the parasite occurs independently of EB1. We overexpressed the MT-binding domain of CLASP1 in parasitized cells. This exhibited a dominant negative effect on host MT stability and led to altered parasite size and morphology, emphasizing the importance of proper MT dynamics for Theileria partitioning during host cell division. Using coimmunoprecipitation, we demonstrate that CLASP1 interacts, directly or indirectly, with the schizont membrane protein p104, and we describe for the first time TA03615, a Theileria protein which localizes to the parasite surface, where it has the potential to participate in parasite-host interactions. IMPORTANCET. annulata, the only eukaryote known to be capable of transforming another eukaryote, is a widespread parasite of veterinary importance that puts 250 million cattle at risk worldwide and limits livestock development for some of the poorest people in the world. Crucial to the pathology of Theileria is its ability to interact with host microtubules and the mitotic spindle of the infected cell. This study builds on our previous work in investigating the host and parasite molecules involved in mediating this interaction. Because it is not possible to genetically manipulate Theileria schizonts, identifying protein interaction partners is critical to understanding the function of parasite proteins. By identifying two Theileria surface proteins that are involved in the interaction between CLASP1 and the parasite, we provide important insights into the molecular basis of Theileria persistence within a dividing cell

Engineering Attenuated Virulence of a Theileria annulata–Infected Macrophage

International audienceLive attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata–transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/cC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata–infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis. Citation: Echebli N, Mhadhbi M, Chaussepied M, Vayssettes C, Di Santo JP, et al. (2014) Engineering Attenuated Virulence of a Theileria annulata–Infected Macrophage. PLoS Negl Trop Dis 8(11): e3183