Regulation of trehalose biosynthesis and thermotolerance by the Cryptococcus neoformans HECT E3 ubiquitin ligase Rsp5

Microorganisms including fungi adapt to profound changes in their local environment during human infections. After exposure to high temperature and other stress conditions, the opportunistic fungal pathogen Cryptococcus neoformans enacts changes in metabolism, cell wall structure, and transmembrane transport that allow it to survive and proliferate in a mammalian host. This stress response program is regulated by the HECT E3-ubiquitin ligase Rsp5 which is required for growth at high salinity, pH and temperature. However, the complete set of Rsp5 substrates that direct these molecular changes remains incompletely understood. Here we demonstrate that C. neoformans Rsp5 confers increased tolerance to temperature and salt stress in part through regulation of the trehalose biosynthesis pathway. Two enzymes in the trehalose biosynthesis pathway, Tps1 and Tps2, are differentially ubiquitinated by Rsp5 after exposure to stress conditions. We directly measured trehalose production after exposure to high temperature and found that a C. neoformans strain lacking Rsp5 is unable to induce trehalose production. Quantitative proteomic analysis of the C. neoformans response to high salinity identified Rsp5-dependent and independent adaptations to osmotic stress, and that Rsp5-dependent ubiquitination does not alter the abundance of Tps1 or Tps2. These results demonstrate that regulation of trehalose biosynthesis is one of the cellular mechanisms by which Rsp5-dependent ubiquitination in C. neoformans facilitates survival in response to stressors encountered in the human infection environment. ImportanceCryptococcus neoformans is an opportunistic fungal pathogen that kills over 180,000 people every year with few effective treatment options. As a yeast that normally lives in the environment, C. neoformans has to survive large changes in its physical environment, including elevated body temperature, when it causes human infections. Here we show how C. neoformans uses a protein modification to regulate production of a fungal-specific metabolic pathway important for survival at human body temperature. Unraveling how environmental fungi tolerate and survive temperature and other stressors will help to understand how they cause disease and identify new and better ways to treat these deadly infections.