Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii.

Little is known about strategies used by pathogens to facilitate CNS invasion. Toxoplasma gondii reaches the CNS by circulating in blood within leukocytes or as extracellular tachyzoites. T. gondii induces EGFR signaling in vitro during invasion of mammalian cells. We examined the effects of endothelial cell EGFR on CNS invasion. Transgenic mice whose endothelial cells expressed a dominant negative (DN) EGFR (inhibits EGFR signaling) exhibited diminished parasite load and histopathology in the brain and retina after T. gondii infection. I.V. administration of infected leukocytes or extracellular tachyzoites led to reduced parasite loads in mice with DN EGFR. This was not explained by enhanced immunity or reduced leukocyte recruitment. Endothelial cell infection is key for CNS invasion. Parasite foci in brain endothelial cells were reduced by DN EGFR. DN EGFR in these cells led to recruitment of the autophagy protein LC3 around T. gondii and spontaneous parasite killing dependent on the autophagy protein ULK1 and lysosomal enzymes. The autophagy inhibitor 3-MA prevented DN EGFR mice from exhibiting reduced CNS invasion. Altogether, EGFR is a novel regulator of T. gondii invasion of neural tissue, enhancing invasion likely by promoting survival of the parasite within endothelial cells.

Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in Schizosaccharomyces pombe.

While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in nonhomologous end-joining (NHEJ) is unclear as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mammals have different requirements for repairing cut DNA ends. Most double-strand breaks (DSBs) require nucleolytic processing prior to DNA ligation. Therefore, we studied repair using the Hermes transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single Hermes insertions using a novel S. pombe transient transfection system, and used Hermes excision to show a requirement for MRN in the NHEJ of nonligatable ends. NHEJ repair was indicated by the >1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had <5 bp of sequence loss or mutation, characteristic for NHEJ and similar excision events in metazoans, and in contrast to the more extensive loss seen in S. cerevisiaeS. pombe NHEJ was reduced >1000-fold in cells lacking each MRN subunit, and loss of MRN-associated Ctp1 caused a 30-fold reduction. An Mre11 dimer is thought to hold DNA ends together for repair, and Mre11 dimerization domain mutations reduced repair 300-fold. In contrast, a mre11 mutant defective in endonucleolytic activity, the same mutant lacking Ctp1, or the triple mutant also lacking the putative hairpin nuclease Pso2 showed wild-type levels of repair. Thus, MRN may act to recruit the hairpin opening activity that allows subsequent repair.