<i>Cryptococcus neoformans</i> is a fungal pathogen that causes cryptococcal meningitis, mainly in immunocompromised individuals, such as those with HIV. Its recent detection on the International Space Station raises concerns about potential health risks in space, where immune systems may be compromised. However, its behavior in space-like conditions remains unclear. In this study, we examined the effects of simulated microgravity on <i>C. neoformans</i>. We found that the condition enhanced the fungus's resistance to membrane and osmotic stress and increased key virulence factors, including capsule formation, melanin production, and urease activity. Using <i>Caenorhabditis elegans</i> as a host model, infections under simulated microgravity were more pathogenic. These findings highlight the potential for increased fungal virulence in space and underscore the need to understand microbial risks for astronaut health and safety in long-term space missions.IMPORTANCEFungi have long been recognized for their remarkable ability to adapt to a wide range of environmental conditions, including extreme environments, such as space habitats. Understanding how fungal organisms, especially pathogenic fungi, adapt to these harsh conditions is crucial for gaining insight into their tolerance mechanisms and the potential emergence of virulence. Our research demonstrates that the pathogenic fungus <i>Cryptococcus neoformans</i> not only survives in space-like conditions but also exhibits increased stress tolerance, enhanced expression of key virulence factors, and elevated pathogenicity in animal models. These findings carry significant practical implications because concerns about fungal contamination in space or other extreme environments may be heightened by the potential for fungi to develop increased virulence through natural adaptation. Our research demonstrates that the pathogenic fungus Cryptococcus neoformans not only survives in space-like conditions but also exhibits increased stress tolerance, enhanced expression of key virulence factors, and elevated pathogenicity in animal models. These findings carry significant practical implications because concerns about fungal contamination in space or other extreme environments may be heightened by the potential for fungi to develop increased virulence through natural adaptation. neoformans , aiming to deepen our understanding and develop strategies for managing this organism both in space and on Earth. RESULTS Simulated microgravity condition does not affect the growth phenotype of C. neoformans The 3D clinostat (Gravite, Space Bio Laboratory) was developed to create a simulated microgravity environment on Earth to mimic the conditions in space flights and the International Space Station (ISS) ( 22 ). This machine provides the simulated micro-weight or microgravity conditions by continuously rotating both axes at a constant speed ( Fig. 1A ). Fig 1 Simulated microgravity conditions do not affect the growth of C. neoformans . ( A ) Left, the 3D clinostat machine was used to simulate the microgravity conditions in this study. Right, the diagram represents the plates continuously rotating both axes in the same direction. ( B ) Simulated microgravity increases membrane and osmotic stress tolerance in C. neoformans Previous research has reported the effects of space exposure on ISS, radiation, and microgravity on the stress response of different microbial species, including fungi ( 25 – 27 ). However, the response to abiotic stresses of C. neoformans has never been described. In this study, we investigate the effects of SMG on multiple stress tolerances, including membrane, osmotic, oxidative, and pH stresses in C. neoformans . Different degrees of stress were set based on the maximum and minimum levels that cells can tolerate in normal growth conditions. The tolerance to membrane stress of C. Although there was a slight increase in colony growth under microgravity simulated by clinorotation, we observed that this effect was not as strong as those seen in membrane and osmotic stress tolerance ( Fig. 2D ; Fig. S1D ). From the above findings, we conclude that SMG conditions enhance membrane and osmotic stress tolerance in C. neoformans , while demonstrating minimal effects on oxidative stress and nearly no effect on pH stress tolerance. Microscopy and growth assays compare fungal strains under normal gravity and simulated microgravity. Results show differences in cell size and capsule thickness, melanin production with L DOPA, and urease activity with urea supplementation. Next, we examined the melanin formation of C. neoformans under SMG conditions. Melanin is an important virulence attribute that has been reported to be involved in the fungal dissemination from the lung to the brain ( 48 ). neoformans pathogenesis. This model organism was chosen because yeast, including cryptococcal species, can serve as the sole food source for C. elegans ( 52 , 53 ), allowing us to observe the pathogenic mechanisms of yeast following ingestion by the worm. The transparent nature of C. neoformans colonies also facilitates monitoring of C. elegans survival using a dissecting microscope. Additionally, C. elegans is typically maintained on solid media, such as nematode growth medium (NGM) agar, making it feasible for incubation in the rotating environment of a clinostat machine during simulated microgravity experiments. The simplest mechanistic link is altered sterol biology—increased ergosterol abundance and/or changes in sterol organization—that can simultaneously stiffen or stabilize membranes under ionic/osmotic conditions while simultaneously increasing the accessibility of AMB binding sites ( 33 , 34 , 56 ). This interpretation is consistent with biophysical reports from artificial and cellular systems showing microgravity-associated increases in membrane fluidity and changes in microviscosity and lipid packing ( 57 – 61 ). Membrane remodeling in C.