Biology subjects

Goich, D.

Publications and source records attributed to Goich, D..

6 recordsLinked to original sources

bioRxiv2026

Yck2 links mitochondrial function and pH homeostasis to cell wall remodeling and antifungal susceptibility in Cryptococcus neoformans

Invasive fungal infections are on the rise due to climate change, antifungal resistance, and increased usage of immunomodulating therapies. The antifungal arsenal is limited in number and in efficacy necessitating novel therapeutics. Deletion of the gene encoding the fungal yeast casein kinase, YCK2, has pleiotropic effects, impacting morphology, drug resistance, metabolism, cell wall, and virulence. Yck2 has recently been shown to be druggable, however the mechanisms by which Yck2 elicits its impacts are unknown. We show that C. neoformans Yck2 is a regulator of cell wall masking, thermotolerance, general stress, and drug resistance. Proximity labeling of Yck2-interacting proteins revealed that Yck2 interacts with proteins that function in the mitochondria as well as the essential plasma membrane H+ ATPase, Pma1. A role for Yck2 in mitochondrial regulation was supported by sensitivity to mitochondrial inhibitors and increased mitochondrial ROS production in the absence of Yck2, and presence of Yck2 in mitochondrial fractions. Using pHluorin-expressing cells we also found that intracellular pH is elevated in cells lacking Yck2 supporting a role in regulating Pma1 activity. Both mitochondria and cellular pH may contribute to cellular signaling and may explain the multitude of phenotypes associated with the yck2{Delta} mutant. Our results begin to identify the mechanisms by which Yck2 contributes to fungal cellular homeostasis which may support efforts to optimize Yck2 inhibitors.

microbiology↗

bioRxiv2026

Stress-responsive roles of the C. neoformans human-like eIF3 complex

Eukaryotic translation initiation factor 3 (eIF3) is a complex of proteins that plays a pleiotropic role in translation regulation across eukaryotes, but the composition of eIF3 complexes varies with retention and loss of subunit genes across evolution. The model yeast Saccharomyces cerevisiae encodes six eIF3 subunits whereas mammals encode thirteen subunits. The basidiomycete fungus and opportunistic fungal pathogen, Cryptococcus neoformans, encodes a mammalian complement of eIF3 subunits. In this report, we investigated the contribution of the non-essential eIF3 subunit genes to cryptococcal stress tolerance. We found that mutants in the four nonessential subunits, eIF3d, eIF3e, eIF3k and eIF3l all exhibit sensitivity to mitochondrial perturbation, and that mutants in eIF3d and eIF3e exhibit opposite susceptibilities to the antifungal drug fluconazole and the hypoxia mimetic cobalt chloride. Loss of eIF3d resulted in reduced eIF2 phosphorylation in response to stress, but the mutant was still able to repress translation to the same extent as the wild type and was defective in induction of integrated stress response regulon. Despite producing higher levels of urease and melanin, the eIF3d deletion mutant was avirulent in Galleria mellonella larvae. Together our data demonstrates the importance of C. neoformans eIF3 in stress adaptation and pathogenesis. ImportanceCryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis in immunocompromised individuals leading to [~]120,000 deaths worldwide annually. When C. neoformans is exposed to host-relevant stressors, such as oxidative stress and thermal stress, it reprograms the translating pool of mRNAs to favor stress adaptation. Eukaryotic translation initiation factor 3 is a multi-subunit complex with roles in stress-responsive translation across eukaryotes yet is unexplored in C. neoformans. We found that C. neoformans encodes orthologues of all thirteen mammalian eIF3 subunits. Mutational analysis of non-essential subunits implicated eIF3 in responses to mitochondrial stress and antifungal susceptibility in C. neoformans, and demonstrates a role for eIF3d in the induction of the integrated stress response as well as in Cryptococcal pathogenesis. Further work will investigate the specific mRNAs that are regulated by eIF3 in response to host-relevant stressors in C. neoformans.

microbiology↗

bioRxiv2025

Hog1/p38 regulates the integrated stress response (ISR) and global transcriptomics changes in C. neoformans during oxidative stresschanges in C. neoformans during oxidative stress.

Cryptococcus neoformans is an opportunistic fungal pathogen that is responsible for an estimated 112,000 deaths per year. We studied the role of the Hog1/p38 and Gcn2 signal transduction pathways during adaptation of C. neoformans to oxidative stress, a physiologically relevant stressor during infection. Using a combination of molecular assays and RNA-sequencing analysis in Hog1-, Gcn2-and double-deleted mutants, we identify shared targets of these pathways, and investigate a negative feedback loop that regulates induction of the integrated stress response (ISR). We found that simultaneous loss of Hog1 and Gcn2 profoundly impacts the ability of C. neoformans to tolerate oxidative stress. We also show that Hog1 regulates induction of the ISR, and that the Hog1 and Gcn2 pathways converge on repression of abundant, pro-growth mRNA, a key step in translatome reprogramming. Our results establish Hog1 and Gcn2 as key regulators of the response to oxidative stress, and demonstrate that the interplay between these pathways is critical for ROS tolerance in C. neoformans. More broadly, our results demonstrate the extent to which signaling via Hog1/p38 and Gcn2 are linked, and point to extensive recalibration of the cellular signaling apparatus when one of these pathways is disrupted. Author SummaryCryptococcus neoformans is a fungus that causes deadly infections, resulting in approximately 112,000 deaths per year. In this study, we examined two genes that are important for the ability of C. neoformans to tolerate stressors it would encounter in the human body: Hog1 and Gcn2. We found that Hog1 and Gcn2 perform overlapping functions during the fungal response to stress. These findings are important because they highlight the potential benefits of targeting these pathways in conjunction to treat infection with C. neoformans. These findings could be used to develop new drug or treatment strategies to reduce the disease burden associated with this infection.

genetics↗

bioRxiv2024

Gcn2 rescues reprogramming in the absence of Hog1/p38 signaling in C. neoformans during thermal stress.

The fungus Cryptococcus neoformans is an opportunistic pathogen of people that reprograms its translatome to facilitate adaptation and virulence within the host. We studied the role of Hog1/p38 in reprogramming translation during thermal stress adaptation, and found that this pathway acts on translation via crosstalk with the Gcn2 pathway, a well-studied regulator of general translation control. Using a combination of molecular assays and phenotypic analysis, we show that increased output from the Gcn2 pathway in a Hog1 deletion mutant is associated with rescue of thermal stress adaptation at both molecular and phenotypic scales. We characterize known outputs of the Hog1 pathway during thermal stress as either Gcn2-dependent or Gcn2-independent, and demonstrate that Hog1 activation regulates the Gcn2 pathway even in the absence of thermal stress. Finally, we implicate this phenomenon in another Hog1-regulated process, morphogenesis, and recapitulate Hog1-Gcn2 crosstalk in the distantly related fungal pathogen, Candida albicans. Our results point to an important link between the stress response machinery and translation control, and clarify the etiology of phenotypes associated with Hog1 deletion. More broadly, this study highlights complex interplay between core conserved signal transduction pathways and the utility of molecular assays to better understand how these pathways are connected. ImportanceCryptococcus neoformans is an opportunistic pathogen of people that causes deadly cryptococcal meningitis, which is is responsible for an estimated 19% of AIDS-related mortality. When left untreated, cryptococcal meningitis is uniformly fatal, and in patients receiving the most effective antifungal regimens, mortality remains high. Thus, there is a critical need to identify additional targets that play a role in adaptation to the human host and virulence. This study explores the role of the stress response kinases Hog1 and Gcn2 in thermoadaptation, which is pre-requisite for virulence. Our results show that compensatory signaling occurs via the Gcn2 pathway when Hog1 is deleted, and that disruption of both pathways increases sensitivity to thermal stress. Importantly, our study highlights the insufficiency of using single gene deletion mutants to study gene function, since many phenotypes associated with Hog1 deletion were driven by Gcn2 signaling in this background, rather than loss of direct Hog1 activity.

microbiology↗

bioRxiv2022

Contributions of Ccr4 and Gcn2 to the translational response of C. neoformans to host-relevant stressors and Integrated Stress Response induction

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, eIF2 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.

molecular biology↗

bioRxiv2020

Glucan unmasking identifies regulators of temperature-induced translatome reprogramming in C. neoformans

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{degrees}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 {beta}-(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.

microbiology↗