Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion

Abstract

The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents.

A Zebrafish Model of Cryptococcal Infection Reveals Roles for Macrophages, Endothelial Cells, and Neutrophils in the Establishment and Control of Sustained Fungemia

“…1I). These results are consistent with previous reports using the H99 strain in zebrafish larvae (36). While inoculated yeast could lodge temporarily in the brain vasculature in the very first hours after inoculation (data not shown), invasion into the parenchyma was not seen until after the establishment of secondary fungemia.…”

“…Inocula of between 150 and 250 cryptococcal cells per larva (estimated mass of 50.5 g at 48 hpi [52]) produce an inoculum-to-mass ratio averaging 3.96, comparable to that of previously published mouse infections (1 ϫ 10 5 yeast cells or spores in an ϳ22-g mouse ϭ 4.54). This dosing was also comparable to that for previously published studies in zebrafish (35,36). For imaging and cryptococcal enumeration experiments, the inoculum for each fish was assessed by direct visual counting via epifluorescence microscopy.…”

“…2B), as were yeast cells ( Fig. 2C) (reported previously for yeast [36]). A small percentage of cryptococcal cells was not associated with either macrophages or neutrophils.…”

“…In contrast, yeast cells of the hypervirulent H99 strain killed the larvae at moderate inoculum of 100 to 200 CFU (Fig. 1B), as shown previously (36). To discern differences between spore and yeast infections, we assessed survival and replication by manually counting the number of Cryptococcus cells every 24 h after inoculation.…”

“…Two recent studies using the larval zebrafish as a model host for cryptococcal infection have demonstrated significant parallels with mammalian pathogenesis and highlighted the unique advantages this organism has to offer (35,36). The zebrafish is genetically tractable and is now well established as a model host for several forms of infection (37)(38)(39)(40).…”