Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii

The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (～70-kDa) TgCK displayed a low affinity for choline (K(m) ～0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by ～60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.     

Limited Impact of 6-Mercaptopurine on Inflammation-Induced Chemokines Expression Profile in Primary Cultures of Enteric Nervous System

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn’s disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.

     

Dipetalonema viteae: resistance in Meriones unguiculatus with multiple infections of stage-3 larvae

The jird, Meriones unguiculatus, infected with 80 normal infective larvae of Dipetalonema viteae, revealed a recovery rate of 27.9% 12 weeks after infection. A pretreatment by three injections of 50 normal larvae each and challenge by 80 larvae resulted in a recovery rate of 10.7%. The recovered worms were longer than those from the challenge control animals. When three times 50 irradiated larvae (35 krad) were inoculated, the recovery rate of the challenge decreased to 2.6%, representing a protection of 90.7%. The surviving adult worms were stunted and derived exclusively from the 80 normal larvae given for challenge, since absolutely no adult worms were recovered in eight animals inoculated three times with 50 irradiated larvae only. Sera of all pretreated jirds contained IgG and IgM antibodies which bound in immunoblotting experiments bound predominantly to three proteins of larvae with molecular masses of 68,140, and 165 kDa, respectively. Enzymatic surface iodination revealed that the three antigens were exposed on the larval surface. The coincidence of a partial resistance to a challenge infection and of an antibody response against surface proteins of infective larvae suggests an importance of these antigens for the rejection of D. viteae mediated by an acquired immunological resistance of M. unguiculatus.