Biology subjects

Panepinto, J. C.

Publications and source records attributed to Panepinto, J. C..

7 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↗

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↗

bioRxiv2022

Arginine methylation of Puf4 drives diverse protein functions

The evolutionarily conserved Pumilio domain-containing RNA binding proteins (RBPs) are involved in many steps of post-transcriptional gene regulation, including RNA stability, polyadenylation, deadenylation, and translation. RBPs are post-translationally modified by methylation of arginine/glycine-rich domains, though the consequences of these modifications are not well known. We determined the arginine methylation and phosphorylation landscape of the Pumilio domain-containing RBP Puf4 from the basidiomycete fungus Cryptococcus neoformans. We found that methyl-deficient Puf4 mutants do not complement critical PUF4 deletion phenotypes, such as resistances to endoplasmic reticulum stress and antifungals, and cell wall remodeling. Methyl-deficient mutants also exhibit unique RNA and protein interactions. Lastly, we identified intra-protein cross talk between post-translationally modified methylated and phosphorylated residues. Overall, we show that post-translational modifications, particularly arginine methylation, of Puf4 regulate the functions of this RBP.

molecular biology↗

bioRxiv2022

The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5{Delta} mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5{Delta} mutant showed that Rmt5 is critical for the homeostasis of eIF2 and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5{Delta} clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5{Delta} phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA-binding proteins. SignificanceThe fungal pathogen Cryptococcus neoformans is a huge threat for people living with immune deficits, especially HIV/AIDS. Its virulence potential is dependent on virulence factors, stress adaptation, and thermotolerance. Post-transcriptional gene regulation is important for these pathogenic processes, but the mechanisms that govern post-transcriptional regulator function are unexplored. Protein arginine methylation is a major modification of post-transcriptional regulators that has not been investigated in pathogenic fungi. Here we investigated the role of arginine methylation by arginine methyltransferases on the biology and virulence of C. neoformans. Phenotypic characterization of deletion mutants revealed pleiotropic functions for RMTs in this pathogen. Further investigation of the Rmt5 interactome using proximity-dependent biotinylation revealed interactions with RNA binding proteins and translation factors, thereby impacting virulence-associated processes.

microbiology↗

bioRxiv2020

Puf4 Mediates Post-transcriptional Regulation of Caspofungin Resistance in Cryptococcus neoformans

Echinocandins have been on the market for 20 years, yet they are the newest class of antifungal drugs. The human fungal pathogen Cryptococcus neoformans is intrinsically resistant to the echinocandin antifungal drug caspofungin, which targets the {beta}-1,3-glucan synthase encoded by the FKS1. Analysis of a C. neoformans puf4{Delta} mutant, lacking the pumilio/FBF RNA binding protein family member Puf4, revealed exacerbated caspofungin resistance. In contrast, overexpression of PUF4 resulted in caspofungin sensitivity. The FKS1 mRNA contains three Puf4-binding elements (PBEs) in its 5 untranslated region. Puf4 binds with specificity to this region of the FKS1. The FKS1 mRNA was destabilized in the puf4{Delta} mutant, and the abundance of the FKS1 mRNA was reduced compared to wild type, suggesting that Puf4 is a positive regulator FKS1 mRNA stability. In addition to FKS1, the abundance of additional cell wall biosynthesis genes, including chitin synthases (CHS3, CHS4, CHS6) and deacetylases (CDA1, CDA2, CDA3) as well as a {beta}-1,6-glucan synthase gene (SKN1) was regulated by Puf4 during a caspofungin time course. The use of fluorescent dyes to quantify cell wall components revealed that the puf4{Delta} mutant had increased chitin content, suggesting a cell wall composition that is less reliant on {beta}-1,3-glucan. Overall, our findings suggest a mechanism by which caspofungin resistance, and more broadly, cell wall biogenesis, is regulated post-transcriptionally by Puf4. ImportanceCryptococcus neoformans is an environmental fungus that causes pulmonary and central nervous system infections. It is also responsible for 15% of AIDS-related deaths. A major contributor to the high morbidity and mortality statistics is the lack of safe and effective antifungal therapies, especially in resource-poor settings. Yet, antifungal drug development has stalled in the pharmaceutical industry. Therefore, it is of importance to understand the mechanism by which C. neoformans is resistant to caspofungin in order to design adjunctive therapies to potentiate its activity toward this important pathogen.

microbiology↗

bioRxiv2019

Translational Regulation Promotes Oxidative Stress Resistance in the Human Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans is one of the few environmental fungi that can survive within a mammalian host and cause disease. Although many of the factors responsible for establishing virulence have been recognized, how they are expressed in response to certain host derived cellular stresses is rarely addressed. Here we characterize the temporal translational response of C. neoformans to oxidative stress. We find that translation is largely inhibited through the phosphorylation of the critical initiation factor elF2 by a sole kinase. Preventing elF2 mediated translational suppression resulted in growth sensitivity to hydrogen peroxide (H2O2). Our work suggests that translational repression in response to H2O2 partly facilitates oxidative stress adaptation by accelerating the decay of abundant non-stress related transcripts while facilitating the proper expression of critical oxidative stress response factors. Carbon starvation, which seems to induce translational suppression that is independent elF2, partly restored transcript decay and the expression of the critical oxidative stress response transcript Thioredoxin Reductase 1 (TRR1). Our results illustrate translational suppression as a key determinant of select mRNA decay, gene expression, and subsequent survival in response to oxidative stress.\n\nImportanceFungal survival in a mammalian host requires the coordinated expression and downregulation of a large cohort of genes in response to cellular stresses. Initial infection with C. neoformans occurs at the lungs, where it interacts with host macrophages. Surviving macrophage derived cellular stresses, such as the production of reactive oxygen and nitrogen species, is believed to promote dissemination into the central nervous system. Therefore, investigating how an oxidative stress resistant phenotype is brought about in C. neoformans furthers our understanding of not only fungal pathogenesis but also unveils mechanisms of stress induced gene reprogramming. We discovered that H2O2 derived oxidative stress resulted in severe translational suppression and that this suppression was necessary for the accelerated decay and expression of tested transcripts. Surprisingly, compounding oxidative stress with carbon starvation resulted in a decrease in peroxide mediated killing, revealing unexpected synergy between stress responses.

microbiology↗