Biology subjects

PANEPINTO, J. C.

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

3 recordsLinked to original sources

bioRxiv2026

NMD-mediated control of Tor influences adaptation to nutrient and temperature conditions in Cryptococcus neoformans

The yeast Cryptococcus neoformans is an opportunistic human pathogen capable of surviving within various environmental conditions. The repertoire of antifungal agents effective in treating cryptococcal infection is limited, necessitating the identification of alternative treatment strategies. Nonsense-mediated decay (NMD) is an RNA decay mechanism that serves as a post-transcriptional regulator of gene expression. While the absence of NMD in C. neoformans sensitizes cells to the antifungal fluconazole, the mechanism underlying this sensitivity and role of NMD in C. neoformans biology remained unexplored. Using phenotypic analysis and RNA-sequencing analysis, we identify basal dysregulation of thermal- and nutrient-adaptive genes and demonstrate temperature- and/or nutrient-dependent phenotypic suppression of upf1{Delta} phenotypes, including fluconazole sensitivity and resistance to rapamycin. We determine rapamycin co-treatment also suppresses the upf1{Delta} fluconazole sensitivity, implicating dysregulation of Tor signaling in phenotypic outcomes when NMD is absent. We then investigate Tor-sensitive signaling in the upf1{Delta} mutant, finding inhibition of cell wall integrity (CWI) signaling and hyperactivation of the kinase Gcn2, both of which returned to wildtype-like levels by either rapamycin treatment, nutrient limitation, or constitutive thermal stress. These results indicated NMD is required for appropriate regulation of Tor signaling in unstressed conditions and suggested upf1{Delta} phenotypes are driven in part by Tor hyperactivation. A phenotypic screen of mutants lacking Tor regulators revealed that deletion of the Tor-suppressing IML1 gene recapitulates upf1{Delta} phenotypes and signaling defects, consistent with Tor hyperactivation. Taken together, our results suggest NMD participates in the regulation of Tor signaling in C. neoformans. Future work will investigate how specific targets of NMD impact Tor signaling and promote fluconazole sensitivity in C. neoformans. ImportancePulmonary and central nervous system infections cause by Cryptococcus neoformans are responsible for about 112,000 deaths annually. Ten-week mortality remains high at 25% with use of frontline antifungals which imposes major health risks due to inherent toxicity. Thus, a need arises to identify novel avenues of treatment, including ways of boosting the efficacy of widely available antifungals such as fluconazole against C. neoformans. The design of NMD inhibitors is an active pharmaceutical pipeline for use in treating human genetic diseases. Even though NMD is conserved across eukaryotes, underlying components and regulatory roles of NMD differ between humans and fungi. Therefore, understanding NMD within C. neoformans will inform the design and repurposing of NMD inhibitors to enhance the antifungal activity of fluconazole as a treatment for Cryptococcosis.

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↗

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↗