Date Published: April 4, 2013
Publisher: Public Library of Science
Author(s): Tong-Bao Liu, Jong-Chul Kim, Yina Wang, Dena L. Toffaletti, Eliseo Eugenin, John R. Perfect, Kee Jun Kim, Chaoyang Xue, Robin Charles May.
Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.
Cryptococcus neoformans is a basidiomycetous yeast pathogen that often causes life-threatening infections. It causes the most common fungal infection of the central nervous system (CNS) in HIV-infected persons and may present as encephalitis, meningitis, or cerebral-space-occupying lesions , , , , , . Cryptococcal CNS infections are uniformly fatal in the absence of treatment , . A recent survey suggests that each year there are around 1 million new cases of cryptococcal meningitis, which result in over 600,000 deaths annually . Despite its medical importance and significant research efforts , , , , the molecular basis of cryptococcal CNS infection and host factors affecting disease development are poorly understood, which complicates efforts for rapid diagnosis and effective treatment. Hence, there is an urgent need to understand the molecular basis of cryptococcal CNS infection to allow the discovery and development of safer and more effective antifungal drugs.
The mechanisms for the frequent occurrence of Cryptococcal CNS infection remain unclear. In this study, we explored our hypothesis that the high abundance of inositol in human brain contributes to virulence of Cryptococcus and the development of cryptococcal meningitis. In our in vitro model of BBB using the HBMEC monolayer, we observed that inositol promotes an increase in Cryptococcus association with and transmigration through the HBMEC monolayer. This increase is dependent upon fungal inositol transporters (ITRs), demonstrated by the fact that mutation of two major ITRs, ITR1A and ITR3C, partially abolishes the stimulation of Cryptococcus transmigration by inositol. Our results indicate that inositol plays a role in the traversal of Cryptococcus across the BBB, and showed that fungal cells can respond to inositol availability in the brain. Because we measured the transmigration rate by counting yeast cell numbers in the bottom chamber in our in vitro model, one concern in interpreting our results is whether addition of inositol to the bottom chamber has an effect on the proliferation of yeast cells. We measured the proliferation rate of Cryptococcus cells in HBMEC medium with or without addition of inositol, and observed a similar growth rate in all strains tested, confirming that the difference in cell numbers in the bottom compartment reflected a difference in yeast transmigration. Cryptococcus can use inositol as a carbon source although it is not a preferred source; Cryptococcus grows very slowly on medium containing inositol as the sole carbon source. Glucose is a much better carbon source for Cryptococcus. The HBMEC medium is enriched in nutrients and contains sufficient glucose for optimal fungal growth, which would explain why inclusion of 1 mM inositol did not affect cell growth. In addition, the association assays demonstrate that inositol promotes the association of Cryptococcus with the HBMEC monolayer. Because a better association often leads to increased transmigration, this effect could explain the increase in transmigration. In addition, because inositol is highly abundant in human and animal brains, for example, the astrocytes that directly interact with the BBB contain over 8 mM inositol that can be rapidly released , , we believe the inositol level in the brain side of the BBB is high and the inositol concentrations we used in in vitro assays are physiologically relevant.