Cell density-dependent regulation of growth and virulence in Cryptococcus neoformans

Abstract

Quorum Sensing (QS) is a communication system used by microbes to coordinate gene expression in response to changes in population density. QS is mediated by secreted molecules that accumulate during cell growth and, after reaching threshold concentrations, induce changes in microbial gene expression triggering population cooperation. In this thesis, we report the existence of QS in the pathogenic fungus Cryptococcus neoformans, the etiologic agent of cryptococcosis, a life-threatening systemic mycosis. Using conditioned medium (CM) we observed significant cell-density dependent effects on C. neoformans growth, glucuronoxylomannan release, melanin synthesis and biofilm formation, important virulence traits in this pathogen. Using different chromatographic strategies, mass spectrometry and nuclear magnetic resonance we found that these effects are produced by a small, stable and very hydrophilic molecule. Even though the molecule was not unambiguously identified, we found that purified CM samples contained pantothenic acid and that commercial pantothenic acid was able to induce activity, although not to the same extent that C. neoformans CM did. These results suggest that a pantothenic add derivative could be involved in QS in C. neoformans, although further experiments need to be carried out to confirm this hypothesis. To understand how this cell-cell communication occurs, we screened a library of approximately 1,200 C. neoformans mutants to identify those that were either unable to produce active CM or failed to respond with increased growth in presence of wild-type CM. We obtained four mutants with the desired phenotypes. Among those mutants, a potential stomatin-like protein, two enzymes of the phosphatidylcholine biosynthesis pathway and the catalytic subunit of the cAMP dependent protein kinase (PKA). These results provide genetic evidence for the existence of intercellular communication in C. neoformans and suggest that the PKA pathway and phospholipid synthesis are involved in this process. In summary, we report the existence of a QS system regulating various virulence traits of C. neoformans. We believe that the study of QS regulation in this fungus will not only improve our understanding its physiology, but also has potential to improve the therapy against cryptococcosis.