In toto imaging shows intravascular proliferation promotes transmigration of daughter cells during brain invasion of Cryptococcus neoformans

Cryptococcus neoformans causes lethal meningoencephalitis. Although the mechanism of its blood-brain barrier (BBB) transmigration has been intensively studied, the predominant route remains unclear. We developed fTRACE, a whole-organ imaging workflow enabling in toto visualization of individual fungal cells in vivo, and found that C. neoformans can proliferate intravascularly before BBB transmigration. Importantly, daughter cells, not mother cells, predominantly transmigrate across the BBB. Intravascular proliferation depends on de novo purine biosynthesis. We created a non-proliferative fluorescent mutant to trace fungal fate in the brain and found that the majority of non-proliferative C. neoformans remained intravascular after prolonged brain infection, while the remainder induced local capillary pruning. Angiophagy occurred with low frequency. C. neoformans can be washed out from the brain vasculature, which is expedited by VEGFR-2 inhibition. This study elucidates a major brain invasion route of C. neoformans and provides an approach for studying host-pathogen interactions using in toto imaging.