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Ergosterol, the major sterol in fungal membranes, is critical for defining membrane fluidity and regulating cellular processes. Although ergosterol synthesis has been well defined in model yeast, little is known about sterol organization in the context of fungal pathogenesis. We identified a retrograde sterol transporter, Ysp2, in the opportunistic fungal pathogen Cryptococcus neoformans . We found that the lack of Ysp2 under host-mimicking conditions leads to abnormal accumulation of ergosterol at the plasma membrane, invagination of the plasma membrane, and malformation of the cell wall, which can be functionally rescued by inhibiting ergosterol synthesis with the antifungal drug fluconazole. We also observed that cells lacking Ysp2 mislocalize the cell surface protein Pma1 and have abnormally thin and permeable capsules. As a result of perturbed ergosterol distribution and its consequences, ysp2 ∆ cells cannot survive in physiologically relevant environments such as host phagocytes and are dramatically attenuated in virulence. These findings expand our knowledge of cryptococcal biology and underscore the importance of sterol homeostasis in fungal pathogenesis. Cryptococcus neoformans is an opportunistic fungal pathogen that kills over 100,000 people worldwide each year. Only three drugs are available to treat cryptococcosis, and these are variously limited by toxicity, availability, cost, and resistance. Ergosterol is the most abundant sterol in fungi and a key component in modulating membrane behavior. Two of the drugs used for cryptococcal infection, amphotericin B and fluconazole, target this lipid and its synthesis, highlighting its importance as a therapeutic target. We discovered a cryptococcal ergosterol transporter, Ysp2, and demonstrated its key roles in multiple aspects of cryptococcal biology and pathogenesis. These studies demonstrate the role of ergosterol homeostasis in C. neoformans virulence, deepen our understanding of a pathway with proven therapeutic importance, and open a new area of study.

Cryptococcus neoformans is a fungal pathogen with significant impact on global health. Cryptococcal cells inhaled from the environment are deposited into the lungs, where they first contact the human immune system. The interaction between C. neoformans and host cells is critical because this step of infection can determine whether the fungal cells die or proliferate within the human host. Despite the importance of this stage of infection, we have limited knowledge of cryptococcal factors that influence its outcome. In this study, we identify cryptococcal genes that affect uptake by both human and mouse cells. We also identify mutants with altered capsule, a protective coating that surrounds the cells to shield them from the host immune system. Finally, we characterize the role of one gene, SAC1 , in these processes. Overall, this study contributes to our understanding of how C. neoformans interacts with and protects itself from host cells.

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of death in immunocompromised individuals. The interactions of this yeast with host phagocytes are critical to disease outcome, and C. neoformans is equipped with an array of factors to modulate these processes. Cryptococcal infection begins with the deposition of infectious particles into the lungs, where the fungal cells deploy various antiphagocytic factors to resist internalization by host cells. If the cryptococci are still engulfed, they can survive and proliferate within host cells by modulating the phagolysosome environment in which they reside. Lastly, cryptococcal cells may escape from phagocytes by host cell lysis, nonlytic exocytosis, or lateral cell-to-cell transfer. The interactions between C. neoformans and host phagocytes also influence the dissemination of this pathogen to the brain, where it may cross the blood-brain barrier and cause an often-fatal meningoencephalitis. In this review, we highlight key cryptococcal factors involved in various stages of cryptococcal-host interaction and pathogenesis.

Ergosterol, the major sterol in fungal membranes, is critical for defining membrane fluidity and regulating cellular processes. Although ergosterol synthesis has been well defined in model yeast, little is known about sterol organization in the context of fungal pathogenesis. We identified a retrograde sterol transporter, Ysp2, in the opportunistic fungal pathogen Cryptococcus neoformans. We found that the lack of Ysp2 under host-mimicking conditions leads to abnormal accumulation of ergosterol at the plasma membrane, invagination of the plasma membrane, and malformation of the cell wall, which can be functionally rescued by inhibiting ergosterol synthesis with the antifungal drug fluconazole. We also observed that cells lacking Ysp2 mislocalize the cell surface protein Pma1 and have thinner and more permeable capsules. As a result of perturbed ergosterol distribution and its consequences, ysp2Δ cells cannot survive in physiologically-relevant environments such as host phagocytes and are dramatically attenuated in virulence. These findings expand our knowledge of cryptococcal biology and underscore the importance of sterol homeostasis in fungal pathogenesis.Competing Interest StatementThe authors have declared no competing interest.

is an environmentally-acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. may be engulfed by these phagocytes, an important step of infection that leads to outcomes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of these two screens, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. The combination of our three screens yielded 45 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence. is a fungal pathogen with significant impact on global health. Cryptococcal cells inhaled from the environment are deposited into the lungs, where they first contact the human immune system. The interaction between and host cells is critical because this step of infection can determine whether the fungal cells die or proliferate within the human host. Despite the importance of this stage of infection, we have limited knowledge of cryptococcal factors that influence its outcome. In this study, we identify cryptococcal genes that affect uptake by both human and mouse cells. We also identify mutants with altered capsule, a protective coating that surrounds the cells to shield them from the host immune system. Finally, we characterize the role of one gene, , in these processes. Overall, this study contributes to our understanding of how interacts with and protects itself from host cells.

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or “CnoCAS9”) reliably enabled genome editing in the widely-used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

There is growing concern that global environmental change might exacerbate the ecological impacts of invasive species by increasing their per capita effects on native species. However, the mechanisms underlying such shifts in interaction strength are poorly understood. Here, we test whether ocean acidification, driven by elevated seawater pCO2, increases the susceptibility of native Olympia oysters to predation by invasive snails. Oysters raised under elevated pCO2 experienced a 20% increase in drilling predation. When presented alongside control oysters in a choice experiment, 48% more high-CO2 oysters were consumed. The invasive snails were tolerant of elevated CO2 with no change in feeding behaviour. Oysters raised under acidified conditions did not have thinner shells, but were 29–40% smaller than control oysters, and these smaller individuals were consumed at disproportionately greater rates. Reduction in prey size is a common response to environmental stress that may drive increasing per capita effects of stress-tolerant invasive predators.

Rising atmospheric carbon dioxide (CO ₂) conditions are driving unprecedented changes in seawater chemistry, resulting in reduced pH and carbonate ion concentrations in the Earth’s oceans. This ocean acidification has negative but variable impacts on individual performance in many marine species. However, little is known about the adaptive capacity of species to respond to an acidified ocean, and, as a result, predictions regarding future ecosystem responses remain incomplete. Here we demonstrate that ocean acidification generates striking patterns of genome-wide selection in purple sea urchins (Strongylocentrotus purpuratus) cultured under different CO ₂ levels. We examined genetic change at 19,493 loci in larvae from seven adult populations cultured under realistic future CO ₂ levels. Although larval development and morphology showed little response to elevated CO ₂, we found substantial allelic change in 40 functional classes of proteins involving hundreds of loci. Pronounced genetic changes, including excess amino acid replacements, were detected in all populations and occurred in genes for biomineralization, lipid metabolism, and ion homeostasis—gene classes that build skeletons and interact in pH regulation. Such genetic change represents a neglected and important impact of ocean acidification that may influence populations that show few outward signs of response to acidification. Our results demonstrate the capacity for rapid evolution in the face of ocean acidification and show that standing genetic variation could be a reservoir of resilience to climate change in this coastal upwelling ecosystem. However, effective response to strong natural selection demands large population sizes and may be limited in species impacted by other environmental stressors.

Introduction: The COVID-19 pandemic disrupted routine childhood vaccinations schedules, posing significant challenges among underserved communities. Understanding how different sociodemographic groups in Tennessee perceive and navigate childhood vaccination barriers is critical for developing strategies to improve vaccination rates and reduce vulnerability to vaccine-preventable diseases. Methods: A cross-sectional survey was conducted to explore barriers to vaccination across diverse sociodemographic groups in Tennessee. Data were collected from caregivers/parents of children aged 18 years and younger across all 95 counties in Tennessee at community events and through partnerships with schools and other local organizations. Parental responses were analyzed to identify barriers in access, concern, and importance domains. The distribution of barriers across different sociodemographic groups such as race, income, education level, and insurance status was identified. Descriptive statistics, non-parametric tests, and log-binomial regressions were used to address the research objectives. Results: This study found that the most prominent barriers to childhood vaccination were concerns regarding vaccine safety and side effects. Significant differences in vaccine barriers were observed across racial and ethnic groups for access barriers (p < 0.001), concern barriers (p = 0.006), and importance barriers (p < 0.001). Parents with lower education levels, children without health insurance, and lower-income families faced disproportionate challenges across two of the three barrier domains studied (access and perceived importance of vaccines). Additionally, concern barriers (aPR = 0.998, p < 0.001) and importance barriers (aPR = 0.997, p < 0.001) were strongly associated with the parent-reported prevalence of up-to-date vaccination status. Conclusions: Addressing parental vaccination barriers related to concern, access, and perceived importance is crucial, particularly for underserved populations including low-income families, uninsured parents, racial/ethnic minorities, and those with limited education. A sustained, equity-focused approach integrating scientific communication, community engagement, and policy interventions is essential for increasing vaccine uptake and ensuring equitable vaccination access.