The cell walls of fungi are critical for cellular structure and rigidity but also serve as a major communicator to alert the cell to the changing environment. In response to stresses encountered in human hosts, pathogenic fungi remodel their cell walls. Masking the β-1,3-glucan component of the cell wall is critical to escape detection by innate immune cells. We previously demonstrated that β-1,3-glucan is unmasked in response to host temperature stress when translatome reprogramming is defective in Cryptococcus neoformans. Here, we used β-1,3-glucan unmasking as an output to identify signaling modules involved both in masking and in translatome reprogramming in response to host temperature stress. We reveal that the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway is involved in translatome reprogramming and that mutants in this pathway display moderate unmasking when grown at 37°C. Additionally, we show that mutants of the cell wall integrity (CWI)/Mpk1 MAPK pathway extensively unmask β-1,3-glucan. While the CWI pathway does not impact translatome reprogramming, our data suggest that it may play a role in the posttranslational regulation of transcription factors that govern masking. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that causes devastating morbidity and mortality in immunocompromised individuals. It possesses several virulence factors that aid in its evasion from the host immune system, including a large polysaccharide capsule that cloaks the antigenic cell wall. Studies investigating how the cell wall is remodeled to keep this pathogen disguised in response to stress have been limited. We previously found that host temperature stress results in translatome reprogramming that is necessary for keeping the highly antigenic β-(1, 3)-glucan component masked. Our data reveal signaling modules that trigger these responses and suggest the points of regulation at which these pathways act in achieving masking. Understanding these mechanisms may allow for therapeutic manipulation that may promote the immune recognition and clearance of this fungal pathogen.

Ets1 is emerging as a key transcription factor that is required to prevent autoimmunity in mice and humans. Ets1 is expressed in both B and T cells, and mice lacking Ets1 are characterized by excess B and T cell activation, leading to enhanced formation of Ab-secreting cells and high titers of autoantibodies. In humans, genome-wide association studies have detected associations of single nucleotide polymorphisms in the human ETS1 gene with autoimmune diseases, including lupus. An increased fraction of CD4 + T cells from Ets1 2/2 mice have an activated effector-memory phenotype, and there are aberrations in differentiation that contribute to the autoimmune phenotype. In vitro studies of B cells suggest that Ets1 may have B cell-intrinsic effects as well. To confirm B cell-intrinsic roles for Ets1, we crossed CD19-Cre mice to mice with a floxed allele of Ets1. Mice with a B cell-specific deletion of Ets1 show increases in B cell activation, numbers of Ab-secreting cells, and levels of autoantibodies, despite the fact that T cells are normal. However, when compared with conventional Ets1 knockout mice, mice with B cell-specific loss of Ets1 have a significantly milder phenotype. These results demonstrate that Ets1 is required in B cells to prevent autoimmune responses but that loss of Ets1 activity in other cell types is required for maximal autoimmune phenotypes. ImmunoHorizons, 2019, 3: 331-340.

Cryptococcus neoformans is a human pathogen capable of causing devastating infections. It must rapidly adapt to changing environments as it leaves its niche in the soil and enters the human lung.

The cell walls of fungi are critical for cellular structure and rigidity, but also serve as a major communicator to alert the cell of the changing environment. In response to stresses encountered in human hosts pathogenic fungi remodel their cell walls. Masking the b-1,3-glucan component of the cell wall is critical to escape detection by innate immune cells. We previously demonstrated that b-1,3-glucan is unmasked in response to host temperature stress when translatome reprogramming is defective in C. neoformans. Here, we used b-1,3-glucan unmasking as an output to identify signaling modules involved both in masking and translatome reprogramming in response to host temperature stress. We reveal that the High Osmolarity Glycerol (HOG) MAPK pathway is involved in translatome reprogramming and that mutants in this pathway display moderate unmasking when grown at 37°C. Additionally, we show that mutants of the Cell Wall Integrity/Mpk1 MAPK pathway extensively unmask b-1,3-glucan. While the CWI pathway does not impact translatome reprogramming, our data suggest it may play a role in the post-translational regulation of transcription factors that govern masking.ImportanceCryptococcus neoformans is a fungal pathogen that causes devastating morbidity and mortality in immunocompromised individuals. It possesses several virulence factors that aid in its evasion from the host immune system including a large polysaccharide capsule that cloaks the antigenic cell wall. Studies investigating how the cell wall is remodeled to keep this pathogen disguised in response to stress have been limited. We previously found that host temperature stress results in translatome reprogramming that is necessary for keeping the highly antigenic β-(1,3)-glucan component masked. Our data reveals signaling modules that trigger these responses and suggest the points of regulations at which these pathways act in achieving masking. Understanding these mechanisms may allow for therapeutic manipulation that could promote immune recognition and clearance of this fungal pathogen.

In response to the host environment, Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host temperature and oxidative stress. This reprogramming is primarily driven through the Gcn2-mediated repression of translation initiation and Ccr4-mediated removal of abundant pro-growth mRNAs from the translating pool. Here we investigate the contributions of these two pathways to the translational response to stress, and show that the response to oxidative stress is primarily driven by Gcn2 whereas temperature and oxidative stress both require Ccr4. Temperature stress, but not oxidative stress, result in an increase in RNase I resistant disome. Further, eIF2α phosphorylation varies in response to the type and magnitude of stress, yet all tested conditions induce translation of integrated stress response (ISR) transcription factor Gcn4, but not necessarily the Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress in C. neoformans. Together this study identifies the differential response to host-relevant stressors in an environmental fungus which can adapt to the environment inside the human host.