Biology subjects

Stuckey, P. V.

Publications and source records attributed to Stuckey, P. V..

6 recordsLinked to original sources

bioRxiv2026

Set202 is a histone lysine methyltransferase that enables Cryptococcus neoformans to adapt to the host environment

All pathogens rely on dynamic gene expression for host adaptation and disease causation. This is especially important for pathogens that normally are found in the environment, like Cryptococcus neoformans, given that environmental and host conditions are dramatically different. Epigenetic modifications, like those carried out by SET-domain histone lysine methyltransferases (HKMTs), are essential for this genome expression flexibility. While transcription factors and signaling cascades have been well-studied in C. neoformanss host adaptation, the role of HKMTs in this process has not been explored. Here, we characterize the gene SET202, previously reported as a putative HKMT important for growth in host lungs. We show that set202{Delta} mutants have 50% less phagosomal permeabilization than the wild-type fungi, have phagosomal maturation defects, and poor intracellular survival. Additionally, they are defective in traits promoting virulence, including significantly smaller capsules, less melanin, and poor growth under host-like conditions. These phenotypes contributed to reduced virulence in a murine model of infection but, notably, this defect was host-temperature-dependent. In order to explain mechanistically how SET202 deletion could affect such a wide range of processes, we assessed and demonstrated, for the first time, that Set202 is a true histone-3-lysine 36 methyltransferase, supporting its role in epigenetically modifying the fungal genome to enable adaptation to the hostile host conditions. Consequently, our findings support the study of the biological function of Set202, as it can serve as a potential future therapeutic target for the treatment of infections caused by C. neoformans, and possibly other pathogenic fungi.

microbiology↗

bioRxiv2025

The vacuolar protein 8 (Vac8) homolog in Cryptococcus neoformans plays conserved and unique roles in vacuolar and cellular morphology, impacting important stress responses and virulence traits

Functionally similar to a plant vacuole or a mammalian lysosome, the fungal vacuole plays a vital role in many cellular processes. Most studies of the vacuole have been performed in the nonpathogenic yeast Saccharomyces cerevisiae; however, recently in pathogenic fungi, the vacuole has been implicated in host invasion in both plants and mammals highlighting an important role for the vacuole in pathogenesis. Here, we report that deletion of Cryptococcus neoformans vacuolar protein 8 (VAC8) results in a fragmented vacuole morphology, impairment of vacuolar fusion, and inability to form titan cells. Additionally, absence of Vac8 results in defective growth at high temperature and in the presence of caffeine, suggesting a defect in cell wall signaling. Interestingly, despite aberrant vacuole morphology, vac8{Delta} is slightly more resistant to fluconazole treatment, and displays increased resistance to hydrogen peroxide, suggesting the irregular vacuole morphology does not impair vacuole function. Like S. cerevisiae Vac8, C. neoformans Vac8 is comprised of armadillo repeat regions which form alpha helices that fold to form a superhelix allowing for increased protein-protein interaction. Many of the known binding partners of S. cerevisiae Vac8 are not present in the C. neoformans genome, suggesting novel functions for Vac8 in this fungus. Notably, deletion of VAC8 affected some virulence traits, providing support to targeting the fungal vacuole as a potential therapeutic intervention.

microbiology↗

bioRxiv2024

The catheterized bladder environment induces dysregulation of macrophage polarization exacerbating bacterial UTI

Urinary catheterization causes bladder damage, predisposing hosts to catheter-associated urinary tract infections (CAUTIs). CAUTI pathogenesis is mediated by bladder damage-induced inflammation, resulting in accumulation and deposition of the blood-clotting protein fibrinogen (Fg) and its matrix form fibrin, which are exploited by uropathogens as biofilm platforms to establish infection. Catheter-induced inflammation also results in robust immune cell recruitment, including macrophages (M{phi}s). A fundamental knowledge gap is understanding the mechanisms by which the catheterized-bladder environment suppresses the M{phi} antimicrobial response, allowing uropathogen persistence. Here, we found that Fg and fibrin differentially modulate M1 and M2 M{phi} polarization, respectively. We unveiled that fibrin accumulation in catheterized mice induced an anti-inflammatory M2-like M{phi} phenotype, correlating with pathogen persistence. Even GM-CSF treatment of wildtype mice to promote M1 polarization was not sufficient to reduce bacterial burden and dissemination, indicating that the catheterized-bladder environment provides mixed signals, dysregulating M{phi} polarization, hindering its antimicrobial response against uropathogens.

immunology↗

bioRxiv2024

Lack of an atypical PDR transporter generates an immunogenic Cryptococcus neoformans strain that drives a dysregulated and lethal immune response in murine lungs

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for >150,000 deaths every year with a mortality rate as high as 81%. This high medical burden is due, in part, to an incomplete understanding of its pathogenesis. In a previous study, we identified a cryptococcal atypical ATP-binding cassette (ABC) pleiotropic drug resistance (PDR) transporter, PDR6, that affected antifungal resistance and host interactions. Here, we follow-up on the role of PDR6 in cryptococcal virulence. In vivo, mice infected with the pdr6{Delta} strain display altered symptomatology and disease progression. Specifically, we observed a significant increase in the innate immune cell populations in the pdr6{Delta}-infected mice when compared to their WT-infected littermates. Furthermore, quantification of pulmonary cytokines/chemokines revealed a robust increase of pro-inflammatory cytokines in mice infected with the pdr6{Delta} mutant strain. Despite the documented sensitivity of the pdr6{Delta} strain to azole antifungal drugs, the treatment of pdr6{Delta}-infected animals with antifungals did not affect survival, yet treatment with a corticosteroid significantly extended survival, highlighting the importance of a balanced/controlled host immune response. Results with mice that mount opposing immune responses supports out hypothesis that the pdr6{Delta} strain induces a hyper-inflammatory immune response, and that the mice succumb to immune-dependent tissue damage rather than the fungal burden. This altered immune response is driven, in part, by changes in the mutants surface. Taken together, this study provides insights regarding cryptococcal pathogenesis and highlights additional functions of PDR-type ABC transporters in pathogenic fungi. IMPORTANCEYeasts of the Cryptococcus genus, especially C. neoformans, can cause disease with unacceptably high mortality. This is due to delays in diagnostics, ineffective treatments, and an incomplete understanding of the interactions between this fungus and our immune system. In this study, we expand our knowledge of the biological function of the PDR6 gene, particularly its effect on modulating the hosts immune response. Normally, C. neoformanss infections are characterized by an anti-inflammatory response that is unable to control the yeast. In the absence of PDR6, the response to the infection is a dysregulated pro-inflammatory response that initially controls the fungi but eventually results in death of the host due to too much tissue damage. This is due, in part, to an altered fungal surface. Given the dual role of PDR6 in modulating antifungal sensitivity and immune responses, this work provides important insights that may lead to new or improved therapeutics.

microbiology↗

bioRxiv2022

Real-time visualization of phagosomal pH manipulation by Cryptococcus neoformans in an immune signal-dependent way

Understanding of how intracellular pathogens survive in their host cells is important to improve management of their diseases. This has been fruitful for intracellular bacteria but it is an understudied area in fungal pathogens. Here we start elucidating and characterizing the strategies used by one of the commonest fungal pathogens, Cryptococcus neoformans, to survive intracellularly. The ability of the fungus to survive inside host cells is one of the main drivers of disease progression, yet it is unclear whether C. neoformans resides in a fully acidified, partially acidic, or neutral phagosome. Using a dye that only fluoresce under acidic conditions to stain C. neoformans, a hypha-defective Candida albicans mutant, and the nonpathogenic Saccharomyces cerevisiae, we characterized the fungal behaviors in infected macrophages by live microscopy. The main behavior in the C. albicans mutant strain and S. cerevisiae-phagosomes was rapid acidification after internalization, which remained for the duration of the imaging. In contrast, a significant number of C. neoformans-phagosomes exhibited alternative behaviors distinct from the normal phagosomal maturation: some phagosomes acidified with subsequent loss of acidification, and other phagosomes never acidified. Moreover, the frequency of these behaviors was affected by the immune status of the host cell. We applied the same technique to a flow cytometry analysis and found that a substantial percentage of C. neoformans-phagosomes showed impaired acidification, whereas almost 100% of the S. cerevisiae-phagosomes acidify. Lastly, using a membrane-damage reporter, we show phagosome permeabilization correlates with acidification alterations, but it is not the only strategy that C. neoformans uses to manipulate phagosomal acidification. The different behaviors described here provide an explanation to the confounding literature regarding cryptococcal-phagosome acidification and the methods can be applied to study other intracellular fungal pathogens.

microbiology↗

bioRxiv2021

Catheterized-bladder environment induces hyphal Candida albicans formation, promoting fungal colonization and persistence.

Catheter-associated urinary tract infections (CAUTIs) account for 40% of all hospital-acquired infections. Given that 20-50% of all hospitalized patients receive a catheter, CAUTIs are one of the most common hospital-acquired infections and a significant medical complication as they result in increased morbidity, mortality, and an estimated annual cost of $340-370 million. Candida spp. - specifically Candida albicans - are a major causative agent of CAUTIs (17.8%), making it the second most common CAUTI uropathogen. Despite this frequent occurrence, the cellular and molecular details of C. albicans infection in the CAUTI microenvironment are poorly understood. Here, we characterize fungal virulence mechanisms and fungal biofilm formation during CAUTI for the first time. We found that the catheterized bladder environment triggers Candida virulence programs and robust biofilm formation through Efg1-dependent hyphal morphogenesis and Als1, an Efg1-downstream effector. Additionally, we show that the adhesin Als1 is necessary for in vitro and in vivo C. albicans biofilm formation dependent on the presence of fibrinogen (Fg), a coagulation factor released in the bladder due to the mechanical damage caused by urinary catheterization. Furthermore, in the presence of Fg, overexpression of ALS1 in C. albicans led to enhanced colonization and dissemination, while deletion of ALS1 reduced both outcomes during CAUTIs. Our study ultimately unveils the mechanism that contributes to fungal CAUTI, which may provide more effective targets for future therapies to prevent these infections.

microbiology↗