The TOR Pathway Plays Pleiotropic Roles in Growth and Stress Responses of the Fungal Pathogen Cryptococcus neoformans

Abstract

The target of rapamycin (TOR) pathway is an evolutionarily conserved signal transduction system that governs a plethora of eukaryotic biological processes, but its role in Cryptococcus neoformans remains elusive. In this study, we investigated the TOR pathway by functionally characterizing two Tor-like kinases, Tor1 and Tlk1, in C. neoformans. We successfully deleted TLK1, but not TOR1. TLK1 deletion did not result in any evident in vitro phenotypes, suggesting that Tlk1 is dispensable for the growth of C. neoformans. We demonstrated that Tor1, but not Tlk1, is essential and the target of rapamycin by constructing and analyzing conditionally regulated strains and sporulation analysis of heterozygous mutants in the diploid strain background. To further analyze the Tor1 function, we constructed constitutive TOR1 overexpression strains. Tor1 negatively regulated thermotolerance and the DNA damage response, which are two important virulence factors of C. neoformans. TOR1 overexpression reduced Mpk1 phosphorylation, which is required for cell wall integrity and thermoresistance, and Rad53 phosphorylation, which governs the DNA damage response pathway. Tor1 is localized to the cytoplasm, but enriched in the vacuole membrane. Phosphoproteomics and transcriptomics revealed that Tor1 regulates a variety of biological processes, including metabolic processes, cytoskeleton organization, ribosome biogenesis, and stress response. TOR inhibition by rapamycin caused actin depolarization in a Tor1-dependent manner. Finally, screening rapamycin-sensitive and-resistant kinase and transcription factor mutants revealed that the TOR pathway may crosstalk with a number of stress signaling pathways. In conclusion, our study demonstrates that a single Tor1 kinase plays pleiotropic roles in C. neoformans.

Zinc Finger Proteins in the Human Fungal Pathogen Cryptococcus neoformans

“…neoformans is confronted with a variety of host-specific stresses, such as nutrient limitation, temperature shift, changes in oxygen/carbon dioxide levels, oxidative/nitrosative stress, and osmotic stress, upon its infection of the host lung. Signaling pathways or factors involved in the stress response have been identified and extensively studied [56,97,98]. ZNFs are also involved in the regulation of the stress response in C. neoformans.…”

Pleiotropy and epistasis within and between signaling pathways defines the genetic architecture of fungal virulence

“…Similar to this past study, we also identified pleiotropic QTGs contributing to more than one virulence-related trait examined here (including thermal tolerance and melanization), namely the genes RIC8 and SSK2. While our study did not share any of the previously implicated QTGs (Lin et al, 2006;Vogan et al, 2016), across several studies -not just those using QTL mapping strategies -observing pleiotropic effects of genes and pathways connected to virulence and virulence-associated traits seems to be a unifying phenomena (Shen et al, 2010;Fang et al, 2012;Tefsen et al, 2014;Lendenmann et al, 2015;Cavalheiro et al, 2018;So et al, 2019;Lev et al, 2019).…”

Interactions of FK506 and Rapamycin With FK506 Binding Protein 12 in Opportunistic Human Fungal Pathogens

“…Although the TOR pathway is a highly conserved mechanism across eukaryotes, its role in C. neoformans is a topic of debate (So et al, 2019). Researchers have found two Tor-like kinases, Tor1 and Tlk1, in C. neoformans (So et al, 2019).…”

“…This study discovered that TLK1 is a dispensable gene resulting in the cultivation of a viable strain following its deletion. The opposite was concluded when TOR1 was deleted (So et al, 2019). Further research explored the function of Tor1 by creating strains that overexpress the TOR1 gene (So et al, 2019).…”

“…The opposite was concluded when TOR1 was deleted (So et al, 2019). Further research explored the function of Tor1 by creating strains that overexpress the TOR1 gene (So et al, 2019). It was concluded that Tor1 negatively regulates two crucial virulence factors, DNA damage response, and thermotolerance by reducing Rad53 and Mpk1 phosphorylation, respectively (So et al, 2019).…”