In toto imaging reveals intravascular proliferation promotes transmigration of daughter cells during brain invasion of Cryptococcus neoformans
Cryptococcus neoformans causes lethal cryptococcal meningoencephalitis. Although the mechanism of blood-brain barrier (BBB) transmigration has been widely studied, the predominant route remains unclear due to technical limitations. Here, we developed a whole-organ imaging workflow, termed fTRACE, that enables in toto visualization of individual fungal cells within host tissue. Further labeling of host counterparts revealed in situ host-pathogen interaction in detail. We utilized fTRACE to address the major route of fungal brain invasion and found C. neoformans can proliferate intravascularly within capillaries prior to BBB transmigration. Notably, by distinguishing mother and daughter cells in vivo, we discovered that daughter cells, rather than mother cells, predominantly transmigrate via the paracellular route. We also identified key factors supporting fungal proliferation in serum as mimicry of intravascular environment. RNA sequencing revealed that serum-growing fungi exhibit a markedly altered gene expression profile. In particular, de novo purine biosynthesis is essential for serum and intravascular proliferation. A fluorescent strain deficient in purine synthesis allowed us to trace the fate of non-proliferating fungi during prolonged brain infection. Unlike inert polystyrene beads, fungal cells showed significantly reduced wash-away events from capillaries - a process enhanced by VEGFR inhibition. For those fungi remain in the brain, the majority remained trapped in capillaries, while a smaller portion was able to induced local capillary pruning. Angiophagy, presumably by transcellular pathway, was detectible but at a low frequency. Collectively, this study not only elucidates the major route of brain invasion by C. neoformans, but also set a paradigm for studying host pathogen interactions using in toto imaging approach. Overall design: RNA-seq profiling of C. neoformans grown in mouse serum versus rich medium(Sabouraud dextrose broth) for 12 hours.